HISTOPATHOLOGY LEC

LECTURE 9: IMPREGNATION AND EMBEDDING PT 1-3

FRANCIS IAN L, SALAVER, RMT, MD
JUNE 28, 2021
For updates and corrections → @mar4rii on Twitter

- Preparing thin sections is not easy bcs the thin sections ● The remaining tissue block can be stored in the laboratory
that you produce will still be thick enough not to allow the for years
light to reach the examiner’s eye from the light source. ○ Purpose of keeping: Patients who wants to have a
- To prepare thin sections with the recommended thickness, second opinion and fot cases wherein the
we will use microtome. pathologist misplaced the slide
- Tissues should be immersed in a chemical that will provide
it a firm consistency Embedding
- That’s why after clearing, issues have to be infiltrated with ● Also known as Casting or Blocking
paraffin so that it will give the tissue firm consistency to be ● Process by which the impregnated tissue is placed into a
able to make thin sections. precisely arranged position in a mold containing a medium
which is then allowed to solidify
INTRODUCTION ● The medium used to infiltrate the tissue is usually the same
● The tissues, after fixation, dehydration and clearing medium utilized for embedding
process, are not sufficiently hard to cut into thin sections
without a suitable support

● Immersing the tissue in melted paraffin

● LEFT = normal gross appearance of one of the lobes of the

lungs
● RIGHT = from a chronic smoker
○ Cigarettes have chemicals that can damage the
lung tissue, leaving behind hollow cavities
○ If the lung tissue is not infiltrated with paraffin, the
hollow cavities will collapse ● After performing infiltration or impregnation, the
○ Thus, we will have a hard time making thin medtech/histotechnologist/pathologist will position the
sections tissue while waiting for the paraffin wax to solidify
● This procedure is referred to as embedding
Impregnation/Infiltration ● At the end of the embedding, u will now have the formation
● Process whereby the clearing agent is completely removed of tissue block
from the tissue and is replaced by a medium that will ● That’s why this procedure is also known as blocking
completely fill all the tissue cavities, thereby giving a firm ● What is the purpose of positioning the tissue during
consistency to the specimen and allowing easier handling embedding step
and cutting of suitably thin sections without any damage to
the tissue and its components

Purposes of Impregnation
● Remove clearing agents
● Fill up the spaces or cavities in tissues for firm consistency
● To produce firm consistency for easy cutting
● To store processed tissues

● LEFT = normal appendix

● RIGHT = erupted or inflamed appendix

● Tissue blocks infiltrated with paraffin

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 Inflamed appendix Paraffin

● The pathologist will do the gross examination first

● Next, cut the appendix with a knife to open its lumen ● Long hydrocarbon chain
● Has carbon and hydrogen
● Since there is no presence of double bonds in the structure
of paraffin, u can classify it as an alkane
● But for an alkane to be considered as paraffin. It must have
more than 16 carbons
● Alkane ka nalang PARAF-FINISH na ang life goals ko!

Paraffin Wax
● Solid at room temperature
● Simplest, most common and best embedding medium used
for routine tissue processing
● Thin sections (4-6 um) may be cut with east from majority
of tissues without distortion
● Paraffin is relatively hard thus sections can easily be cut
● Serial sectioning is easy to do
● Upon the examination, the pathologist will find the presence
of abundant neutrophils within the tissue of the appendix Serial sectioning
● Serial sectioning is defined as obtaining a continuous
ribbon of sections from a paraffin block and placing all the
sections on multiple slides

● Remember when we need to come up with the cross

section of the appendix, In order of the pathologist to
diagnose
● That’s why during embedding procedure, the
medtech/histotech will position the cut section of the
appendix this way (last pic right) because it is only in this
position that we can come up with the cross section of the
appendix
● Thus, it is important to do embedding, blocking, or casting.

● We can create ribbon or series of thin sections and have all

of them processed in the lab

Paraffin Wax
● Process is very rapid, allowing sections to be prepared
within 24 hours (from dehydration to staining)
● Ideal for routine tissue processing
● Tissue blocks and unstained mounted tissue sections may
be stored in paraffin in an indefinite period of time after
impregnation without considerable tissue distortion
● If the cut sections of the appendix were position properly, it ● Many staining procedures are permitted with good results
will be easy for us to come up with the this section showing ● Does not interfere with majority of staining procedures
the cross sections of the appendix utilized in histopath laboratory
Disadvantages:
4 general types of Tissue impregnation and Embedding media ● Remember: paraffin is solid at room temperature and for it
● Paraffin wax to infiltrate the tissue, the paraffine should be in liquid form
● Celloidin ● Overheated paraffin can cause tissue to shrink and to
● Gelatin become brittle
● Plastic ○ Pag masyado siyang HOT, nagiging MARUPOK
ka
Take note: Whatever medium u will use for the tissue impregnation ● Lymph nodes and nerves are lost
or infiltration, it will also be the same that u will use for embedding ○ Because they are small
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 ● Maintain temperature to 2-5 degree above melting point of ● Take note: the medium used in infiltration is the same
wax chemical used in embedding
○ Going beyond this temperature will cause ● During infiltration, the medium should be in a liquid state.
problems with the tissue processing ● During embedding, the medium should solidify
● Prolonged impregnation will cause excessive tissue ● To keep the medium in liquid state during infiltration, the
shrinkage and hardening, making the sections difficult to medium should be in melted.
cut ● Higher melting point means the wax is difficult to melt and
● Inadequate impregnation will cause the retention of therefore, it is a hard type of wax!
clearing agent (xylene is still present) ● Conclusion: The higher melting point, the harder the wax….
○ Tissue becomes soft and shrunken ● The harder the wax, the easier it is to make thin sections…
○ Tissue blocks crumble or break up when floated in ● Hard wax requires heavy duty type of microtome during
a water bath sectioning!!!!
Complete vs Incomplete Infiltration
Sliding and Sledge microtome

- Left: adequately infiltrated = good thin sections ● Commonly available paraffin waxes have the ff melting
- Right: inadequately infiltrated = retention of the xylene; point in:
folded thin sections ○ 45 degree Celsius
○ 52 degree Celsius
Tissue sections break up when floated in a water bath ○ 56 degree Celsius – most commonly used
○ 58 degree Celsius
What makes the paraffin waxes of varying melting points is that,
paraffin is an alkane. As defined on the introduction to paraffin,
paraffin is an alkane that can contain more that 16 carbons, The
varying number of carbons now will have an effect on the melting
points of the paraffin waxes

QUESTION!
● The higher the melting point, the harder the wax
● The harder the wax, the easier to make thin sections

● THEN WHY NOT USED the paraffin wax with the melting
- If inadequately infiltrated, the tissue sections will break up
point of 58 degree Celsius?
when floated on the water bath leading to formation of
cracks in the tissue
ANSWER
● The recommended temperature for infiltration is usually 2-5
Liver
degree celsius above the melting point of the wax to ensure
it is completely melted
● Paraffin with melting point of 56 degree celsius = temp
58-61
● Paraffin with melting point of 58 degree celsius = temp
60-63
○ Higher temperatures can cause tissue to become
brittle and to shrink and these changes will start at
temperatures above 60 degree celsius

- Tissue distortion and cracks due to inadequate infiltration Procedure

● After clearing, the tissue is submerged in 2-4 changes of
Paraffin Wax melted paraffin wax, either in a paraffin oven or in an
incubator regulated at 55-60 degree celsius
● Paraffin processing is not recommended for fatty tissues ● In a room temperature (20-24 degree celsius), paraffin wax
○ Difficult for the paraffin to completely infiltrate with a melting point of 54-58 degree celsius is
brain and breast recommended
○ Fatty tissue (brain, breast) ● If the laboratory temperature is between 15-18 degree
Celsius, the melting point of the wax should be between
50-54 degree celsius

SHORT QUIZ (TRUE OR FALSE)

1. The higher the melting point of paraffin, the harder it is to
cut tissue sections
2. Paraffin waxes with high melting point require heavy duty
microtome during sectioning
3. Overheated paraffin causes tissue shrinkage and
brittleness
4. For a paraffin wax with melting point of 56 degree celsius,
● The dehydrating and clearing agents used in the process the recommended temperature during infiltration should be
dissolve and remove fat from the tissue 61 degree celsius
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ESSAY/ FILL IN THE BLANKS
● 1-3. Three consequences of incomplete infiltration

● 4. In a laboratory with temperature of 15-18 degree

Celsius, the paraffin to be used should have a melting point
between _________ degree Celsius

Three ways of Paraffin wax Impregnation and Embedding

● By manual processing
● By automatic processing - reduce workloads and shorten Oscillating tissue basket
processing time ● The tissue basket oscillates up and down in each station at
● By vacuum embedding three-second intervals to ensure thorough and even mixing
of the reagents and optimum tissue infiltration.
I. MANUAL PROCESSING ○ Also to provide agitation
● After completion of clearing step, we will get the tissue and ■ To enhance paraffin infiltration
put it on top of a solid paraffin placed in a tissue block, and ■ If paraffin infiltration is enhanced by
put both the tissue block and solid paraffin inside an oven agitation in automatic processing, how
set at 56-60 degree celsius many changes of paraffin now should be
○ Heat will cause the paraffin to melt, causing the needed to complete infiltration in
tissue to be submerged in the melted paraffin automatic processing?
(start of infiltration process)
● At least 4 changes of wax are required at 15 minutes
intervals in order to ensure complete removal of the
clearing agent from the tissue.
○ Tissue must be immersed in melted paraffin for 15
mins
○ Tissue is transferred to another solid paraffin,
placed into the oven, then melted again
○ This procedure have to be repeated for 2 more
times
■ A total of at least 4 changes of wax
within 15 mins. interval
● The specimen is then immersed in another fresh solution of
melted paraffin for approximately 3 hours to insure
completeness of embedding or casting (until specimen
solidifies)
10% Buffered 24 hours
FIXATION
Formalin

70% Alcohol 6 hours

95% Alcohol 12 hours

● RIGHT BLUE ARROW - Beaker 1
DEHYDRATION 100% Alcohol 2 hours ○ Inside is the oscillating tissue basket (moves up
and down to mix and agitate)
100% Alcohol 1 hour ○ Contains fixative
○ Left and middle arrows - Beaker 2 and 3
100% Alcohol 1 hour ■ Also contains fixatives
● The automated tissue processor has a total of 12 beakers
Xylene or Toluene 1 hour
CLEARING 10 Beakers or Jars
Xylene or Toluene 1 hour ● Transparent beakers
○ Beaker I – fixative (formalin) 1-2 hours
○ Beaker II – fixative 1 hour
Paraffin Wax 15 minutes
○ Beaker III – fixative 30- 45 minutes
■ Fixation
Paraffin Wax 15 minutes ○ Beaker IV – 70% alcohol. 30 minutes
IMPREGNATION ○ Beaker V – 90% alcohol. 30 minutes
Paraffin Wax 15 minutes ■ Alcohol is used to dehydrate the tissue

Paraffin Wax 15 minutes

EMBEDDING Paraffin Wax 3 hours

AUTOMATIC PROCESSING
● Automated Tissue processor
● Makes use of an automatic tissue processing machine
which fixes, dehydrates, clears and infiltrates tissues
thereby decreasing the time and labor needed during the
processing of tissues resulting to rapid diagnosis with less
technical errors
○ Beaker VI – Absolute alcohol. 1 hour
○ Beaker VII – Absolute alcohol. 1 hour
○ Beaker VIII – Absolute alcohol. 1 hour
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 ■ To complete dehydration step ● There is also 2-3 changes of wax needed to remove the
○ Beaker IX – Xylene. 1-2 hours clearing agent and to impregnate the specimen
■ Clearing step ○ This is made possible due to constant tissue
○ Beaker X – Xylene 45 minutes – 1 hour agitation which accelerates and improves tissue
■ Clearing penetration giving rise to more consistent results

2 Thermostatically controlled beakers Advantages of Automated processing

● Thermostatically controlled beakers ● It’s very efficient
○ Designed for paraffin infiltration (requires heat to ● Saves time and energy to operate
keep paraffin melted) ● Cost effective and user friendly
■ Transparent beakers can't withstand ● Can process different tissues same time
heat ● The machine does the transfer of tissue from one bath to
○ Wax bath I (done at 45°c) 2 hours another.
○ Wax bath II. 2 hours
■ Both contain melted paraffin for Precautions
infiltration ● Presence of any odor of the clearing agent during final
paraffin wax bath indicated that the paraffin should be
changed
● Dehydrating agents should be frequently changed since
dehydration is the most critical stage of tissue processing
and inadequate dehydration is difficult to correct once
tissue is in paraffin
○ It will affect clearing, infiltration and embedding
● Automated tissue processors have 10 transparent beakers,
the first 3 contain the fixative agent while the last 2 contain
the clearing agent, beakers 4,5,6,7,8 contain the
dehydrating agent (TAKE NOTE: We have absolute
● How many changes of paraffin is required for automatic alcohols in beakers 6,7,8)
processing? ○ Beaker VI – Absolute alcohol
○ Only 2 ○ After processing 2 batches of specimens, expect
○ Number is reduced because of constant agitation that the absolute alcohol in Beaker 6 will be
diluted by the water that was drawn out from the
specimens
○ Thus, we need to discard the content of beaker 6
■ Move beaker 7 to the position of beaker
6, followed by beaker 8, and load a new
transparent beaker on the previous
position of beaker 8 with fresh absolute
alcohol in it
○ TAKE NOTE: After processing 2 rounds of
specimen, there’s a need to discard the content of
● Blue arrow is pointed at one of the thermostatically Beaker 6
controlled beakers (designed for paraffin infiltration ● After two complete processing runs of load, the first 100%
alcohol bath (Beaker 6) should be always discarded and
Lifting Mechanism and Electric rotor at the base the other moved down so that the final batch has fresh
absolute 100% alcohol
● The clearing agent and the other diluted alcohols can be
changed at least once a week
● To avoid spillage, fluid and wax containers must be filled to
the appropriate level. Any spillage should be wiped away
● Wax accumulating on the surface of the beaker should be
removed
● Wax bath thermostat should be maintained at least 3
degrees above the melting point of the wax and should be
checked when loading the machine
○ To make sure that the wax is in its liquid state for
complete infiltration

III. VACUUM EMBEDDING

● Method is almost similar to manual processing of paraffin
● Where oscillating baskets are attached to infiltration
● Lifting mechanism is the one responsible for the up and ● Involves the wax impregnation under negative
down movement of the oscillating tissue baskets to promote atmospheric pressure inside an impregnation and
agitation embedding oven to hasten the removal of the air bubbles
● When it’s time for tissue to be transferred to the next and clearing agent from the tissue block thereby promoting
beaker or jar, the cover of the machine is raised up, and the a more rapid tissue impregnation
lifting mechanism carefully removes the tissue basket and ○ Normal atmospheric pressure: 760 mm Hg
gently transfers it to the next beaker. ○ Negative atmospheric pressure - value need not
to be less than 0, as long as the value is lower
II. AUTOMATIC PROCESSING than 760 mm Hg, that is already considered as
● The machine is mounted on rollers to permit turning of negative atmospheric pressure
platforms and easy access to beakers and wax bath ○ Ex. 760 mm Hg will be lowered to 500 mm Hg
● Makes use of 12 individual processing steps with 10 one (negative atmospheric pressure)
liter capacity glass beakers and 12 thermostatically ○ The container where we will perform paraffin
controlled wax baths with a safety device cut-out switch to infiltration is attached to a vacuum
protect wax from overheating
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 ■ Main purpose of vacuum is to suck in air ■ Responsible got sucking out air to lower
so that the pressure in the container will the atmospheric pressure inside the
be lowered down (producing neg heavy brass chamber
atmospheric pressure)
○ Advantages: removing air from the container and
the specimen
■ Presence of air in the specimen will
make it hard for the paraffin to
completely infiltrate it
■ Ex. alveoli of the lungs (filled with air)
● It removes residual bubbles such in the lungs
● Another advantage - vapors of the clearing agent are
immediately removed by the vacuum
● Particularly recommended for urgent biopsies and for
delicate tissues such as lungs, brain, decalcified bones,
eyes, spleen and lymph nodes
○ The tissue is then not over-exposed to heat,
brittleness, shrinkage, and hardening
● Time required is reduced from 25% to 75% of the normal
time required. (Fastest)
○ Therefore among the three ways of doing paraffin
infiltration, vacuum embedding is considered to be
the fastest
● The vacuum chamber is enclosed in a thermostatically
controlled water jacket which is usually maintained at a
temperature 2-4 degree Celsius above the melting point of
the wax
○ Water jacket
■ Main purpose: not to allow heat to
dissipate outside the container so that
the temperature inside the heavy brass
chamber is maintained at 2-4C

● Main purpose of the pump or vacuum is to lower the

atmospheric pressure inside the heavy brass chamber from
760 to 400 to 500mmHg
● Manometer - determines that the pressure is 400-500
● One valve allows readmission of air when the bath is under
negative pressure
● The other valve is connected to a tube which in turn is
connected to a pump, to allow exhaustion of
400-500mmHg.
○ Allows readmission of air

VACUUM EMBEDDING PROCEDURE

● Composed of flat-bottomed heavy brass chamber with 1. Clear the tissue using xylene.
heavy glass lid with thick rubber valves which produces an 2. Place tissue in paraffin wax inside the vacuum chamber
airtight seal when the chamber is being used and make the oven airtight by closing the heavy glass lid
○ Red arrow: and the valves
■ where we will perform the paraffin 3. Exhaust the air slowly by means of vacuum pump until
infiltration there is negative pressure of 400 to 500nmHg
■ Positioned on top of the heating element - Creates negative atmospheric pressure inside the
- the one responsible for providing heat heavy brass chamber
to keep the paraffin in the melted state 4. Leave for 15 minutes then slowly readmit air until normal
■ Covered by a heavy glass lid atmospheric pressure is reached
○ Heavy glass lid - main purpose of which is to not - Has to be done slowly so that the air that will go
allow air from the outside to enter the heavy brass inside the heavy brass chamber will no longer go
chamber to maintain the negative atmospheric inside the spaces inside the tissue
pressure 5. Place the tissue in a fresh wax.
○ Green arrow: rubber valve 6. Repeat steps 3 and 4.
■ Connected to a vacuum 7. Place the tissue in a fresh wax
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 8. Repeat step 3 and leave for 30-45 minutes ○ Free from dust, water droplets and foreign matter
- Take note: firsat wax = 15mins, second change of ● Fresh wax should be filtered at 2 degree Celsius higher
wax = 15mins, third change of wax, 30-45mins than its melting point
9. Bring to normal atmospheric pressure and proceed to ● Wax that has been trimmed away from the impregnated
embedding tissue may be melted and filtered for future use with a
● Slowly readmit air coarse filter paper
● Number of changes of wax = 3
○ Thus, recommended for urgent biopsies TRIMMING OF TISSUE BLOCK
○ Tissue will not be overexposed to heat
● Number of changes of blocks in manual
processing = 4

- Trimmed pritions will now be melted and filtered using a

coarse filter paper so we can still utilize it for another round
of paraffin infiltration
ALWAYS FILTER FRESH WAX

QUIZ TIME
A. VACUUM EMBEDDING
B. MANUAL PROCESSING
C. AUTOMATIC PROCESSING

1. Recommended temperature is 2-5 degree Celsius above

melting point = B
2. Recommended temperature is at least 3 degree Celsius
above melting point = C
3. Recommended temperature is 2-4 degree Celsius above - Presence of cotton fibers because it wasnt filtered
melting point = A - Failure to filter reused or trimmed paraffin will cause
4. Number of paraffin changes is at least 4 = B contamination of tissues from the previous specimen to the
5. Number of paraffin changes is 3 = A specimen that is currently being processed
6. Number of paraffin changes is 2-3 = C
- Only 2 because of constant agitation which
enhances paraffin infiltration
7. Infiltration under negative atmospheric pressure
8. Uses paraffin oven
9. Fastest
10. With constant agitation
11. 12 beakers

Factors Affecting Paraffin Impregnation

● bone, teeth, brain and eye are difficult to infiltrate (BeeBee)
using paraffin
● (its hard to infiltrate the heart of my Beebee crush)
○ Require prolonged period of immersion with
paraffin - Thyroid follicle came from the previous specimen that was
○ REMEMBER: prolonged immersion with paraffin processed using the reused or trimmed paraffin
causes tissue shrinkage and hardening GREEN’S NO 904
○ THUS there are other recommended embedding
medium for these tissues
● Benzene and Xylene are easily removed from the tissues
while chloroform and cedarwood oil requires frequent wax
changes.
○ Adding benzene to cedarwood oil would hasten
the process
○ If the clearing agent used is chloroform or
cedarwood oil, they are difficult to remove thus
they require frequent wax changes
○ If cedarwood oil is used and you want to hasten - Recommended filter paper
the process of paraffin infiltration, you can always - Coarse type
add benzene to the cedarwood oil
Precautions observed in paraffin wax impregnation in General ● If wax is suspected to be contaminated with water, it can be
● Prolonged treatment with paraffin causes tissue shrinkage subjected to 100-105 degree Celsius to remove the water.
and hardening of the tissue, making cutting difficult ● Paraffin wax can only be reused once.
● Overheated paraffin (above 60 degree Celsius) can cause
tissue shrinkage and hardening
● Paraffin wax must be pure
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PARAFFIN EMBEDDING
● Tissue will now be embedded in paraffin wax melted at 5-10
degree Celsius above the melting point
● Cooling is done:
○ Refrigeration at -5 degree Celsius
○ Immersion in cold water
○ Tissue tek with cold plate
TISSUE TEK

● Sold in pellet forms

● Provides easier handling and faster melting characteristics
● Cuts cleanly and smoothly without cooling
● Assures ribbon continuity with sectioning
● Free of foreighn contaminants
○ No need to perform filtration
Embeddol
● Watch: https://www.youtube.com/watch?v=Nab97hfUVPc ● Similar to Paraplast
● Can be use as substitute
Fill in the blanks ● Melting point of 56-58 degree Celsius
1. if paraffin is contaminated with water, the remedy is to
________ Bioloid
2. Trimmed paraffin can be reused once provided that
___________
3. Give 4 tissues that are not easily infiltrated by paraffin
4. three ways on how to solidify paraffin during embedding ● Bioloid
5. how to hasten infiltration with cedarwood oil? ● Semisynthetic wax recommended for embedding eyes
● For organs with thin wall and circular shaped sections
Substitutes for Paraffin Wax
● Paraplast Tissue Mat
● Ester Wax ● Paraffin with rubber
● Water Soluble Waxes ● With same property as Paraplast
Paraplast Ester Wax
● Paraplast is suitable for tissue infiltration. ● Paraffin = higher melting point = harder wax = good for
● Melting point: 56-57 degree Celsius 4-6um thin sections BUT……
○ Similar to the most commonly used paraffin wax ● Has lower melting point than paraffin (46-48 degree
used in the laboratory Celsius) but is harder than paraffin
● Sold as pellets - easier for the histotechs and medtechs to ○ Does not follow the principle of paraffin: the higher
estimate how many pellets should be added into the tissue the melting point, the harder the wax is.
block for paraffin infiltration ● Main component: Diethylene glycol distearate
● Paraffin wax is sold as large block - med techs and ● Good for sections as thin 2-4 microns
histotechs need to cut small pieces of that paraffin blocks to ○ Problem: brittle sections
come up with the estimated about of paraffin wax needed ○ Once this section is placed on the water bath, the
for tissue infiltration tissues can easily disintegrate or break up.
● It is a refined combination of highly ● Remedy: Added with:
purified paraffin with plastic ○ Castor oil
polymers. ○ Ethyl cellulose
● Paraffin plastic polymers = paraplast ○ Stearic acid
● Paraplast – Paraffin plus Plastic!! ■ Most commonly used.
● More elastic and resilient because of ● Ethylene glycol
the plastic component
○ permitting large dense
tissue blocks such as
bones, teeth and brain to
be cut easily with the same
result as in double
embedding.

● Serial sections can be cut with ease

without cooling the block, thereby
preventing formation of ice crystals
○ Ice crystals can damage
cells/tissue and that is avoided in paraplast
because there’s no need for you to cooldown the
block BLUE ARROW: diethylene glycol, above it are 2 (two) ethylene glycol
● It is soluble in common clearing agents and follows the molecules.
same time schedule for paraffin impregnation, and does not ● If these two ethylene glycols molecules will form bond with
tend to crack like other paraffin wax substitutes. each other, they will form diethylene glycol
● What links between the two ethylene glycol is ESTER
BOND pointed by the blue arrow, reason why ester wax is
named diethylene glycol distearate
● Not soluble in water but soluble in 95% ethyl alcohol

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 ○ Not a concern because water will be removed by ○ Solid at room temp= there's a need for us to melt
the dehydration step. to keep it in the liquid state for it to infiltrate the
○ Take note that ester wax is soluble in 95% ethyl tissue
alcohol-- dehydrating agent . Carbowax
○ Infiltrating medium is miscible to dehydrating ● Carbowax is soluble in water hence does not require
agent. dehydration and clearing of tissue
■ That’s why we can skip the clearing ○ After the tissue is fixed, it is washed out to
step. remove excess fixative and precipitates and
○ Tissue can be impregnated without prior clearing transferred directly to melted carbowax
■ But will depend on the protocol in the ○ Processing time is reduced
laboratory. ○ Can skip dehydration and clearing
■ Some laboratories would prefer to
perform clearing step before ● Harmful effects of dehydrating agents and clearing agents
impregnating the tissue with ester wax. are avoided
○ Cellosolve and xylene may be used as clearing ● Fats which are usually removed by dehydrating and
agents (depending on the protocol of the hospital clearing agents are not removed
or preferences of the pathologist) ○ Fats are preserved
Should the laboratory recommend that clearing should be ○ Eating too much CARBs can make you FAT! Fat
done? is not removed from the body!!!Carbowax
● Clearing agent must be gradually removed before you ● Tissues are not exposed to too much heat
proceed with wax impregnation. ○ There is no excessive hardening, shrinkage and
○ Tissue must be placed in a solution containing brittleness of tissue
equal amount of clearing agent and ester wax for ○ Cytologic details are preserved
3-6 hrs before transferring the tissue to pure ester ■ Eating too much CARBs can make
wax youFAT!
○ 3-4 changes of wax should be done to ensure ■ No gym workout = No HEAT
complete tissue impregnation ■ No heat = No shrinkage of the body
■ If you are already performing the size, hardening of muscle.
infiltration step, you need to have 3-4 ● Suitable for enzyme histochemical studies because
changes of wax that is to ensure enzymes are denatured greatly by heat
complete infiltration or impregnation. ○ Since in carbowax tissues are not overexposed to
● Sectioning of Ester wax-impregnated tissue should be done heat then enzymes are most likely preserved
on a heavy duty microtome (sledge or sliding type) due to ● Four changes of carbowax are needed (melted in 56
the relative hardness of the wax degree Celsius)
○ Once in 70% for 30 mins (first change)
○ Once in 95% for 45 mins (second change)
○ Twice in 100% Carbowax for 1 hr each with
agitation (third change)
■ 3rd and 4th changes will involve pure
100% carbowax
○ Specimen is then embedded in carbowax and
rapidly cooled in refrigerator (fourth change)
■ Whatever is our infiltrating medium will
also be our embedding medium.
● Hygroscopic property = easily dissolve in water.
● Tissue block must not have contact with water or ice.
Water-Soluble Waxes ○ If you want to cool down your tissue block inside
the refrigerator make sure that the tissue block
will not come in contact with water or ice.
○ Remedy: wrap tissue blocks in cellophane or
plastic.
● Tissue sections are difficult to float out and mount out due
to its extreme solubility in water
○ Remedy: Add soap in water to reduce distortion
and promote flattening and floating out of sections
○ Or add 10% polyethylene glycol.

Matching type
A. Ester Wax D. Bioloid
B. Carbowax E. Tissue mat
C. Paraplast

If two ethylene glycol molecules together will form ester bond = ester 1. Paraffin with rubber
wax 2. Paraffin with plastics
Chain of ethylene glycols= POLYETHYLENE GLYCOLS 3. Elastic and resilient
● There must be at least 12 ethylene glycols. 4. Dehydrating and clearing can be bypassed
● If lesser than 12, simply refer them as deca,nona, hexa etc. 5. Composed of polyethylene glycol
● polyethylene glycols are made up of more than 12 carbons. 6. Embedding medium recommended for eyes
● Composed of polyethylene glycol with melting points of 7. Low melting but a hard type of wax
38-42 or 45-56 degree Celsius 8. Water-soluble wax
● Carbowax – polyethylene glycol composed of 18 carbons or 9. Composed of diethylene glycol
more which appears solid at room temperature 10. Requires heavy duty microtome.
○ Most popular and commonly used

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 IMPREGNATION AND EMBEDDING PART 2 Celloidin/Collodion
● High viscosity
Disadvantages of Paraffin ○ Can slow on its rate of infiltrating
● Cannot completely infiltrate bone, teeth, brain and eye ○ Thus, it is made available in 3 preparations
● Overheated paraffin can tissue brittleness and shrinkage ● It is supplied in
● Prolonged immersion in paraffin causes tissue shrinkage ○ thin (2%),
and hardening ■ 2-4% of celloidin
■ The purpose of diluting celloidin is to
Celloidin/Collodion lower down its viscosity and enhance its
● Purified form of nitrocellulose soluble in many solvents, ability to infiltrate
suitable for the following specimens ○ medium (4%)
○ Organs with large hollow cavities (eye, lungs) ■ 4-6%
■ Completely infiltrated ○ thick (8%) solutions of cellulose
○ For hard and dense tissues (bone and teeth) ■ 8-12%
■ Easily infiltrated compared to paraffin ● Celloidin is available in powder
○ Delicate tissues (embryos) ● Thus, there is a need to dissolve the powder of celloidin in
a solvent before we can come up with the thin medium and
Nitrocellulose thick preparations
● dissolved in equal parts of ether and alcohol (solvent).
● For example, to prepare 8% celloidin
○ Get 8 grams celloidin.
○ Mix it with 100ml solvent (50 ml ether + 50 ml
alcohol)

Preparation
● The fastest way to dissolve celloidin is to soak it first in half
the final volume of anhydrous ethanol to soften it (50 mL for
each 8 grams celloidin), with intermittent mixing in a tightly
stoppered container. (Leave for 24 hours)
● Cellulose ● The next day, an equal volume (50 mL) of ether is added
○ Considered as polysaccharide because there is a and intermittently mixed until an evenly consistent solution
chain of monosaccharides particularly glucose is obtained
○ The chemical structure of nitrocellulose is similar
to cellulose it’s just that there is a presence of Celloidin/Collodion
nitrogen ● Dense tissues which are hard to infiltrate (e.g. bones and
brains) and specimen which tend to collapse easily due to
air spaces (e.g. eyes and lungs) are supported better,
thereby avoiding the crumbling of tissues during sectioning.
● When eye sections are embedded by the paraffin method,
the retina may be detached from the harder tissues (e.g.
sclera and choroid) that encircle it.
●

● Nitrocellulose
○ This is named such bcs it looks like cellulose but
there is an addition of nitrogen

● YELLOW = Retina
● RED = Choroid
○ Surrounds the retina
● WHITE = Sclera
○ Surrounds the choroid
● If this eye specimen is infiltrated by a paraffin, the yellow
● Whenever we try to get extracts of plants, we observe them colored retina will detach from the choroid and the sclera
to be viscous or syrupy in characteristic
● Expect that nitrocellulose will appear syrupy or viscous

● RED = Retina (innermost)

● Tissue blocks containing celloidin; appears syrupy or ○ Nucleated cells which are responsible for
viscous perceiving light and stimulating the optic nerve
10
 ● YELLOW = Choroid
● BLUE = Sclera Celloidin blocks
● Hard tissue of the eye = Choroid & Sclera
● Soft tissue = Retina

- Very difficult to section (thin and serial sections)

● It permits cutting of tissue sections which are thicker than

paraffin wax, and is therefore recommended for processing
of neurological tissues.
○ Thicker sections contain higher number of
neurons
● LEFT = normal histology
● RIGHT = Retinal detachment
○ Detachment of the retina from the choroid &
sclera

- This is how neurons appear in the microscope

- Neuron pointed by red arrow: appreciate
presence of that neuron because the thickness of
this section is more than 10 micrometer
- Thicker section = higher number of neuron that
● Trauma and diabetes mellitus can cause retinal detachment
you can study from a neurological tissue
● There is a need to use a medium that will not cause retinal
detachment to determine whether its is brought about by a
trauma or paraffin

● Gross appearance of retinal detachment - Rabies is acquired through the bite of an infected
animal
Celloidin/Collodion - Virus enters the peripheral nerve and
● Its rubbery consistency allows tissue blocks that are either transported to the brain and spinal cord
very hard or of varying consistency, to be cut without undue - The involvement of the organs of CNS
distortion will most likely result to the death of the
○ If the sections will crumple in the microtome, u patient
can easily stretch or flatten them out - The autopsy of the patient will require
● It does not require heat during processing; hence, tissue sections of the brain and spinal
producing minimum shrinkage and tissue distortion cord to study them under the
● It is, therefore, recommended in cases when minimum microscope
shrinkage is required and frozen section technique cannot
be done. Negri bodies in neurons of a patient who died from rabies
Disadvantage:
● Celloidin impregnation is very slow (lasting for several days
or weeks)
○ Very slow since it highly viscous
● Very thin sections (less than 10u) are difficult to cut (softer
than paraffin)
● Serial sections are difficult to prepare

- If the sections of the brain and the spinal cord

- You cannot prepare thin sections and serial were impregnated and embedded with celloidin,
sections in celloidin embedded tissues expect that the sections that we will prepare will
- Just imagine yourself cutting a gelatin. be thick
11
 - Higher number of neurons = higher probability to There are two methods for celloidin impregnation of tissue
see the presence of negri bodies ● Wet Celloidin method
- Come up with correct cause of death ● Dry Celloidin method

● Vapor of the ether solvent is very inflammable; hence, it Wet Celloidin Method
should never be used near an open flame. ● Recommended for bones, brain sections and whole
● Solvents are very volatile and therefore must be kept in organs. (Wet BB Wipes)
bottles with ground – glass stoppers to prevent evaporation. ● After the usual fixation and
○ Solvents used to prepare the thin, medium and dehydration of the tissue, it is placed in
thick preparations of celloidin are highly volatile equal parts of ether and alcohol for 12
– 24 hours.
Celloidin as solid is known as gun cotton ○ Clearing can be by passed
● Celloidin itself is highly flammable ○ Celloidin is miscible with
especially if its prepared as a gun alcohol, and since our
cotton dehydrating agent is an
alcohol, there’s no need for
us to remove the alcohol by the clearing step
○ Take note: tissue must be immersed first in a pure
solution of ether and alcohol (does not contain
● https://www.youtube.com/watch?v=KBVBELMq20U any celloidin in it)
■ To prepare it for the actual celloidin
Histology Laboratory impregnation
○ Impregnation with celloidin is more time
consuming that paraffin

Tissue is then placed in thin celloidin (2–4%) for 5–7 days

● Pictures of microscopic images are usually circular because

of the lens

Photomicrography

Transferred to medium celloidin (4–6 %) for another 5–7 days

- Majority of the histology photos we can see on the

internet are rectangular
- Made possible by installing a camera
system into the microscope, this is
referred to as photomicrography Transferred to thick celloidin (8– 12%) for 3-5 days

● Photomicrographs are difficult to obtain.

● Thickness of the section affects the resolution of the image

● The tissue is immersed in equal parts of ether and alcohol

for 12 - 24 hours (1 day)
● The tissue is then placed in thin celloidin (2 - 4%) for 5 - 7
days,
● Transferred to medium celloidin (4 - 6%) for another 5 - 7
- Compare the 2 photos - one has better quality days,
and resolution ● Drained off and poured with thick celloidin (8 - 12%) until
- The photo with the better quality and resolution the specimen has become impregnated, usually between 3
was taken from a tissue embedded with paraffin - 5 days
- RECALL: we can not prepare thin sections if the ● 1 + 7 + 7 + 5 = 20 days!!!!! (slow process)
tissue was embedded with celloidin
- Photomicrographs are difficult to obtain
if our infiltrating and embedding medium
is celloidin
12
Transferred to thick celloidin (8 - 12%) for 3 - 5 days Low Viscosity Nitrocellulose Method
● Take note:
○ Higher concentration means better tissue
penetration
○ Higher viscosity means poor tissue penetration or
infiltration
○ Celloidin = higher concentration means higher
viscosity
■ Higher the concentration of celloidin, the
poorer the tissue penetration is
● Low viscosity nitrocellulose
○ Similar to celloidin because celloidin is also
● Ensure that the container is tightly sealed composed of nitrocellulose
○ What makes it more advantageous than celloidin
Wet Celloidin Method is that is can be made available at a low viscosity
● The thick celloidin preparation is for the actual embedding but with a high concentration
procedure ○ Can penetrate the tissue more rapidly than the
● The specimen is removed from the celloidin, and ordinary celloidin
transferred to an embedding medium containing freshly ● Another form of celloidin soluble in equal concentration of
poured thick celloidin and kept in a tightly covered jar or ether and alcohol with a lower viscosity, allowing it to be
dessicator in order to evaporate the alcohol – ether solvent. used in higher concentrations and still penetrate tissues
● Desiccators - to remove the vapors of the solvents of the rapidly.
celloidin
● We allow the solvents to evaporate, leaving behind pure Low viscosity
celloidin on our tissue block. Pure celloidin will harden - this Preparations Celloidin
Nitrocellulose
is now embedding/casting/blocking
● The dessicator top is removed for a few seconds, time and Thin 2% 5%
again, to admit fresh air and harden tissue block.
○ This is to allow readmission of fresh air so that the Medium 4% 10%
desiccators can get more vapors from the
solvents from the container/jar Thick 8% 15%
● Evaporation must be gradual to achieve a consistent, ● Preparations of both have the same viscosity
uniform degree of hardness throughout the block and ● Conclusion:
prevent the formation of air bubbles. ○ At lower preparations, both celloidin and LVN
● For celloidin embedding to be considered as complete, the have the same viscosity. However, at the same
solvents should have totally evaporated and the celloidin level viscosity, LVN has a higher concentration
black ○ Comparing the thin preparations of celloidin and
● When the ball of the finger leaves no mark on the surface of ○ LVN, the latter has better tissue penetration or
the tissue block, evaporation and consequently, infiltration LVN is also cheaper than celloidin
embedding, is considered to be complete. ○ LVN is superior to celloidin because at a given
○ Use the thumb and not your fingernails!!! viscosity, LVN has a higher concentration
● Prolonged contact with air dries out and hardens celloidin. compared to celloidin.
○ Affects the quality of the specimen that we can ● Celloidin is available as thin (2%) , medium (4%) and thick
prepare during the sectioning step (8%) preparations (lowest viscosity to highest viscosity)
● The tissue block is then stored in 70 – 80% alcohol until ● The equivalent concentrations for LVN are 5%, 10% and
ready for cutting/sectioning. This is done to avoid 20%.
dehydration and shrinkage of tissues. ● Note: at same the viscosity, LVN has a higher
○ Because it is stored in 70-80% alcohol, we refer to concentration. It means it has better tissue penetration
it as WET CELLOIDIN METHOD ● It forms a harder tissue block and makes cutting of thinner
sections possible.
Dry Celloidin Method ● Problems: Sections tend to crack during staining
● Preferred for processing of whole eye sections ○ The tendency of tissues to crack may be
○ DREYE Method!! prevented by adding plasticizers (e.g. Oleum
● The principle and procedure of this method is similar to wet Ricini or Castor oil)
celloidin method, except that 70-80% alcohol is not used for ● More explosive than celloidin and should therefore be
storage before cutting handled with care.
● Gilson’s mixture, which is made up of equal parts of ○ Since LVN has higher concentration than that
chloroform and cedarwood oil, is added to the celloidin celloidin expect that it is more explosive.
block before hardening. ● When dry, striking or dropping the container will cause the
● The cedarwood oil used in the Dry Celloidin Technique substance to explode.
helps to soften the layers of the eye, preventing the retinal ○ It is usually marketed while wet with alcohol.
detachment. ● The container must be kept tightly covered and protected
● Cedarwood oil is added twice daily for 10 days until the from sunlight to avoid evaporation of alcohol.
mixture is composed of 90% percent cedarwood oil. ○ LVN is highly explosive especially when it is dry.
○ Initially there is 50% cedarwood oil and 50% ● When no longer needed for future use, the nitrocellulose
chloroform. should be carefully disposed, since the material becomes
○ The purpose of this method is to make the tissue increasingly dangerous as the alcohol continues to
transparent evaporate.
○ Alcohol is not used in Dry method because it will ○ The more that it will stay inside the laboratory, the
dissolve the cedarwood oil. higher the chances that the alcohol has already
■ Will no longer make the tissue evaporated causing the LVN to dry up and
transparent and it will increase the become highly explosive.
chance of retinal detachment

13
Double Embedding Method Transferred to fresh solution of 20% Gelatin with 1% Phenol
● Peterfyi's celloidin-paraffin wax technique which is then allowed to cool in a refrigerator
● the process in which tissues are first infiltrated with celloidin
and subsequently embedded in paraffin mass.
○ Celloidin is a better infiltrating medium than
paraffin because it can infiltrate organs that
paraffin cannot completely infiltrate
■ Organs: brain, bones and teeth
● Will be subsequently
embedded in paraffin.
● Thin sections are easily made
if the tissue is embedded in
paraffin.
○ This is used to facilitate cutting of large blocks of
dense firm tissues like the brain, bone, and teeth
○ Also recommended for making small sections of
celloidin blocks. Summary of the procedure
○ Appears to be promising, how come we are not ● After the fixative has been completely washed out, the
using it in the laboratory today? tissue is
■ Highly explosive ○ placed in 10% Gelatin with 1% Phenol for 24
■ Problems with paraffin have already hours
been addressed by paraplast. ○ transferred to 20% Gelatin with 1% Phenol for the
next 12 hours, and
Gelatin impregnation ○ finally to another fresh solution of 20% Gelatin
with 1% Phenol which is then allowed to cool in a
● Water soluble; no need for us to remove water using
refrigerator until impregnation and embedding are
alcohol and remove alcohol using the clearing agent.
completed.
● Gelatin impregnation is rarely used except
Gelatin–embedded tissues are then transferred to 10% formalin
○ when dehydration and clearing is to be avoided
for 12 – 24 hours in order to harden the tissue..
because gelatin is water soluble
○ when tissues are to be subjected to histochemical
and enzyme/lipids studies.
● Can skip dehydration and clearing steps.
● lipids are removed by dehydration and clearing agent from
the tissues and since we will not be performing dehydration
and clearing, lipids are preserved.
● Heat is also not used, expect that proteins such as
enzymes are preserved.
● Fixatives (such as 10% formalin) should still be washed out
by running water before tissue is infiltrated by gelatin.
○ Soluble in water so the tissue must be thoroughly
washed with water after fixation to make it easier
for gelatin to infiltrate the tissue.
● It is used as an embedding medium for delicate specimens ● Tissues should not be more than 2 – 3 mm thick since
and frozen tissue sections gelatin–embedded specimens are harder to freeze than
○ because it prevents fragmentation of tough and unimpregnated tissues.
friable tissues when frozen sections are cut. ● “The cold never bothered me anyway”
● Used along with 1% phenol to prevent growth of molds
● Can be used in fresh frozen tissue section. IMPREGNATION AND EMBEDDING PART 3
Placed in 10% Gelatin with 1% Phenol for 24 hours Embedding
● After impregnation, the tissue is placed into a mold
containing the embedding medium and this medium is
allowed to solidify
● This allows hardening of tissues, giving them a firmer
consistency and better support, thereby facilitating the
cutting of sections
● Whatever medium is used to infiltrate the tissue is also the
same medium that will be used to embed the tissue
● Tissues are placed and arranged at the bottom of a mold
and are immersed in melted paraffin at a temperature
between 5-10°C above is melting point and;
○ Infiltration = 2-5°C above melting point bcs
After 24 hours, tissue is then transferred to 20% Gelatin with 1% overheated paraffin can cause the tissue to shrink
Phenol for the next 12 hours and become brittle
○ In embedding, we are also tasked to orient the
tissues and properly position the tissue for cutting
○ For us to have enough time to properly position
the tissue, the paraffin should not solidify
immediately
○ That is only made possible if the paraffin is melted
at a higher temperature
○ Then cooled rapidly
■ Refrigeration at -5°C
■ Immersion in cold water

14
 ○ The are done to cool down the temperature of the COMPOUND EMBEDDING UNIT
paraffin thereby preventing the effect of ● Made up of series of interlocking plates resting on a flat
overheated paraffin to the tissue metal base, forming several compartments
● Orientation ● It has the advantage of embedding several specimens at a
○ Process by which a tissue is arranged in precise time, thereby reducing the time needed for blocking
position in the mold during embedding, on the
microtome before cutting, and on the slide before
staining
○ Generally speaking, the surface of th section to be
cut should be placed
parallel to the bottom of
the mold in which it is
oriented

● The surface of the tissue to be cut

● Shown in the photo (right) are used in fresh frozen section
is parallel to the bottom of the mold
because they are made up of plastic and they will melt with
where it is oriented.
heated paraffin
● Expect that the compound embedding unit used in routine
should be made up of metal
Advantages:
Several types of blocking out molds may be used
● We can embed several specimens at a time
● Leuckhart’s Embedding Mold
○ The time needed for embedding will be reduced
● Compound Embedding Unit
or shortened
● Plastic Embedding Rings and Base mold
● Disposable Embedding Molds
PLASTIC EMBEDDING RINGS AND BASE MOLD
LEUCKHART’S EMBEDDING MOLD ● Consist of a special stainless steel base mold fitted with
a plastic embedding ring which later serves a s the block
● Also known as L mold
holder during cutting
● Consists of two L-shaped strips of heavy brass or metal
arranged in a flat metal plate and which can be moved to
adjust the size of the mold to the size of the specimen
● Two L-shaped strips of heavy brass are placed on top of a
metal plate
● One good thing about it is that u can adjust the size of the
tissue block that u can form

● Used in Tissue Tek

● A machine equipped with a warm plate to manage the
impregnated specimen, and a cold plate at -5C for rapid
solidification of the block
● BLUE ARROW: Warm Plate
○ Where we impregnate the tissue with melted
● The L-mould are kept on a metal plate. Tissue is placed the paraffin
surface of the glass and is oriented ● RED ARROW: Cold plate
● The plate is then filled with molten wax. The wax is then ○ Where we allow the melted paraffin to solidify to
cooled L-moulds are then removed perform the embedding procedure

As soon as u have already adjusted the L-shaped brass to come up

with the recommended size of the tissue block
↓
Place the tissue in the middle of the two strips of heavy brass
- Take note: The surface/side of the tissue that is supposed
to be cut/sectioned should be placed facing the metal plate
↓
Pour the melted paraffin (5-10°C above its melting point)
↓
Wax is allowed to cold own and solidify
↓
L-shaped strips of heavy brass will then be removed from the setup

Advantages:
● Easy to procure and reusable
○ There are a lot of manufacturer that can provide
the lab with this type of embedding mold
● Blocks produced are even, with parallel sides, and with a
fairly shaped initial setting of the wax
● Adjustable to make different size of blocks
● It is recommended for routine use, although too slow and
cumbersome for use in a busy laboratory
Disadvantage:
● U can only process one specimen at a time
○ U need to wait for the paraffin to solidify before u The base mold is filled up with melting paraffin
can remove the L-shaped strips of heavy brass ↓

15
 Advantages of Tissue tek
● Easy of use
● Less paraffin wax is needed
● Faster embedding
○ Only 5 mins to solidify in the cold plate
● Tissue is firmly attached to the holder with
permanent identification
○ Bcs u can always label the plastic
embedding ring with the name or serial
number of the patient
● Resectioning is easily done
The specimen is oriented on the base mold while the paraffin is still ● Blocks can be filed up after sectioning
in its melted form ○ For storage
↓
DISPOSABLE EMBEDDING MOLDS
● Peel-away disposable thin plastic embedding molds
○ Recommended for plastic resin embedding
○ paraffin wax
○ Can be assembled and disassembled
○ During the process of embedding, you should
assemble the embedding mold, place tissue in the
center, and pour embedding medium
○ A disposable embedding mold - only used once

The plastic embedding ring is placed in position

↓

● `Plastic Ice Trays

○ used in ordinary refrigerators may be
recommended for busy routine laboratories.
○ Each compartment may be utilized for embedding
one tissue block, which may then be removed by
bending the plastic tray once the wax has
Fill up the plastic rings with melted paraffin wax
solidified; or by smearing the inner mold with
↓
glycerin or before embedding
The tissue is then allowed to cool down on the cold plate of the
■ Can embed multiple tissue at a time
Tissue tek equipment.
After filling up, transfer the whole set up (the stainless steel base
mold with the tissue inside it and the plastic embedding rings on top
of it) to the cold plate to allow the paraffin wax to solidify for the
embedding procedure
↓
Upon hardening (5 minutes)
Entire setup should stay in the cold plate for 5 mins to ensure
solidification of the paraffin wax and completeness of the embedding
procedure
↓
The tissue is taken out together with the embedding ring and is
immediately ready for cutting without having to undergo trimming ● Paper boats
thereby saving time and effort ○ Utilized for embedding celloidin blocks but are
RECALL: equally useful for paraffin wax blocks.
● Prior to placing the whole set up into the cold plate of the ○ They have the advantage of being cheap and
tissue tek, u filled up the stainless steel base mold and easy to make. They provide easy and accurate
even the plastic embedding rings with melted paraffin identification of specimen, thereby avoiding
● Melted paraffin is overflowing into the plastic embedding confusion and interchange of tissue blocks
rings and solidifies ○ Rapid embedding of small or large volume of
● If u try to separate the plastic embedding rings from individual specimens is possible, since paper
stainless steel base mold, the tissue and the paraffin or the molds can be made to suit any size of tissue.
tissue block will be removed along the plastic embedding
ring and will look like this:

This tissue block is ready for section thereby reducing the time &
effort.
16
QUIZ!! ■ Main purpose of capillaries is to
a. peel-away d. Leuckhart introduce blood into the glomerulus to
b. paperboat e. Compound Embedding unit be filtered
c. plastic ice trays f. Plastic ring/Base mold ● Right picture: Glomerulus under light microscope

1. Use in Tissue Tek - F

2. L-shaped brass - D
3. Blocks are removed by bending the mold - C
4. Cheap and easy to make - B
5. one component acts as the blockholder - F
6. less paraffin is used - F
7. interlocking plates with several compartments - E
8. not ideal for busy laboratories - D

PLASTIC (Resin) Embedding

● Allows cutting of sections thinner than paraffin
○ In paraffin, we can prepare sections as thin as
4-6um
○ In plastic (resin), we can prepare thinner than 4-6 ● Cut section of one of the capillaries in the glomerulus
um ○ Has 3 layers that play important roles in the
● Provides superior results for light microscopic studies process of filtration
● Recommended for high resolution microscopy such as ● BLUE ARROW: Fenestrated Capillary (First layer)
electron microscope ○ Fenestrated because it has pores
○ Take note: In electron microscope, we can study ■ Medical term for pores = fenestra
even the subcellular details of the tissue, and for ○ Fenestra will allow small substances (including
that to be possible, tissues should be as thin as proteins) to pass through it leaving behind blood
possible cells -- this is why you shouldn't see blood cells in
● Renal biopsies the urine because even with the presence of
● Bone marrow biopsies fenestra, its size isn't enough for blood cells to
pass through
Kidney biopsies (Nephron) ● RED ARROW: Basement membrane
○ Has a negative charge that repels negatively
charged proteins
○ Even if proteins have passed through the
fenestrated capillary, they will not go out through
the urine because they will be repelled back to the
blood by the negatively charged basement
membrane
● YELLOW ARROW: Cell called podocyte
○ Podocyte have processes extending from its
body, these processes will wrap around the
capillary
● Major processes will give rise to smaller processes and
these processes will have spaces in between them.
● Basic functional unit of the kidney (Nephron) ● Spaces are provided with tight junctions - main purposes of
○ Most dilated portion - glomerulus which is to limit smaller proteins to pass through as well
■ Primary site for the filtration of blood to ● Urine should be free of proteins because the large proteins
remove waste products and form urine were repelled by the basement membrane and the small
proteins were not allowed to pass through by the filtration
slits or the spaces between the processes of the podocytes
which are rich in tight junctions
● To appreciate the layers of the capillary: use plastic or resin
as embedding medium

Glomerulus under ordinary light microscopy:

● Glomerulus
○ Blood passes through it so that blood will undergo
filtration
■ Filtration will remove all the waste
products in the blood and eliminate
through urine

● Glomerulus is globular in shape

○ Center: group of capillaries

17
Using electron microscope: ● Left photo:immune complexes deposited on the
fenetstrared capillary of the glomerulus
● Since these material are considered as foreighn to the
glomerulus, they will be considered as antigens and will be
attacked by the immune system
● Immune system will send neutrophils towards the area
where the immune complexes are deposited
● Immuno complexes will be cleared through phagocytosis of
neutrophils. But, in the process of phagocytosis, some of
the enzymes of the neutrophil will leak out and destroy the
surrounding tissue
● This results to the destruction of the glomerulus brought
about by the enzymes leaking out from the neutrophils

● Blue arrows: fenestrated capillary of the glomerulus

● E: endothelium
○ Blood vessels are lined by simple squamous
epithelium ● Left photo: normal glomerulus
● F: fenestrae of the fenestrated capillary of the glomerulus ● Right photo:
● Red arrows: basement membrane directly coating or ○ Neutrophils inside trying to eliminate the
surrounding the fenestrated capillary deposited immune complexes
● Yellow arrows: secondary or small processes of the
podocyte
● You cannot appreciate the fenestrated capillary in the
glomerulus if paraffin wax is used and studied under light
microscopy
● All of these structures are best studied under electron
microscopy and embedded with plastic or resin

● Observable under light microscopy: abundance of

neutrophils in the glomerulus

Electron microscope view of one of the capillaries in the

glomerulus showing the presence of neutrophil in its lumen:

● There are conditions that can cause antibodies and

antigens to form immune complexes.
● Immune complexes are usually removed from the body by
the action of macrophages
● If macrophages and monocytes are overwhelmed by the
number of immune complexes, these immune complexes
now will be deposited to several tissues in the body
○ These tissues include the joints, skin, blood
vessels, and glomerulus of the kidney

18
BONE MARROW BIOPSIES ○ Araldite (bisphenol A) – slowest, large molecule
with high viscosity
■ Has the largest size and has the highest
viscosity.
■ Least efficient,
○ Glycerol-based (epon) – faster, smaller size than
araldite, lower viscosity but cannot be prepared in
pure form
■ It has smaller size than araldite and
lower viscosity.
■ Problem: it cannot be prepared in pure
form.
○ Cyclohexene dioxide (spurr) – low viscosity and
can obstained in pure form
● Cell on the left: Myeloblast ■ The most efficient among the three
○ Can give rise to the mature white blood cells that ● Hydrophobic – not compatible with most water soluble
we see in our peripheral blood smear stains.
● Pro-normoblast: most immature stage of red blood cells ○ Majority of the dyes we use are water soluble.
● These 2 cells can be distinguished from each other using ● Irritation on skin and airways.
electron microscope: Take note: Gloves must be worn while processing and
processing should be done inside fume hoods to eliminate
toxic vapor and avoid irritation to the airways
● Contains vinylcyclohexene dioxide – carcinogenic - skin
cancer.
● Reduces antigenicity – not recommended for
immunohistochemical studies.
○ Immuno- there's involvement of antibodies and
antigens.
○ Example: Cytokeratin 18 is overexpressed on the
surface of cells in colon cancer.

● Myeloblast on the left side

○ Presence of the granules
Plants embedded using plastic or resin:

Cytokeratin 18 overexpressed in Colon cancer Cells

WATCH: https://www.youtube.com/watch?v=CEPpQlr0p5M

EPOXY PLASTICS ● Should these cells become malignant because there's

● Composed of catalysts and accelerators colon cancer?
● A catalyst will enhance or hasten the hardening of the ○ These cells now will start to express Cytokeratin
plastic embedding medium without it being consumed in the 18 on their surfaces.
reaction (similar to enzymes) ● For us to identify whether the cut section of the colon or the
● Accelerators on the other hand also hasten the hardening specimen presented to the laboratory we have to simply
of the plastic embedding medium but are consumed during identify whether the cells are expressing cytokeratin or not.
the reaction
● 3 types of epoxy plastics: Plastic covers the antigen of specimens
○ Araldite (bisphenol A)
○ Glycerol-based (epon)
○ Cyclohexene dioxide (spurr)
● Efficiency is based on size of molecules, viscosity and
purity
○ Large molecules will find it hard to infiltrate and
embed the tissue
○ If they have low viscosity
■ The higher the viscosity of the
embedding medium the slower that it
will infiltrate the tissue
○ Should be prepared in a pure form
● Which among the three is the most efficient?
● Composed of Epoxy plastics, catalysts and accelerators
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The next thing that we will do is to add antibodies directed against ACRYLIC PLASTICS
the cytokeratin 18, but we will make sure that the antibodies carry ● Composed of acrylic or methacrylic acid for light
along with them an enzyme or a fluorescent die. microscopy
● Enzyme now will impart color to the cells if the cells are ● Advanatges:
expressing cytokeratin 18. ○ Less viscous than plastic resins = less time
needed for embedding
Cytokeratin 18 overexpressed in Colon cancer ○ Hydrophilic- compatible with majority of our water
soluble dyes
● Polyglycol methacrylate (GMA)
○ Hydrophilic – compatible with water soluble
stainsAcrylic Plastics
● Methyl methacrylate (GMA)
○ Preferred for its relative hardness = better
sectioning
○ Preferred over glycol methacrylate
● Problem: Prematurely polymerize in the presence of light,
that’s why acrylics should be stored in amber(dark) bottle

Quiz !!!
A. Epoxy
B. Acrylics
Antibodies have already attached themselves to the expressed 1. Lesser time needed for embedding- B
cytokeratin 18 of the cancer cells. ● Lower viscosity and better tissue penetration.
● Antibodies are labeled with enzymes, this will impart color 2. Can cause skin cancer- A
to the cells. 3. Can reduce antigenicity of the tissue-A
● The presence of colored changes will give us an idea that 4. Has a special requirement for storage B
the cells are expressing cytokeratin 18 and therefore the 5. airway and skin irritant A
patient has colon cancer. 6. Not compatible with most dyes A
Cytokeratin 18 overexpressed in Colon cancer cells 7. recommended for renal and bone marrow biopsies A

A. Spurr
B. Epon
C. Bisphenol A
1. Slowest acting
2. Faster than araldite but cannot be prepared in pure form
3. Fastest and can be prepared in pure form
4. Also known as Glycerol-based epox

Left: normal histology of the colon; Right: similar cells that are
stained with brown color.
● The presence of the brown staining color will tell the
pathologist that the antibodies identified the presence of
cytokeratin 18 on the cells submitted to the lab.

PROBLEMS WITH EPOXY PLASTICS

● They tend to cover antigens expressed by the cancer cells

○ Tend to reduce antigenicity of the target cell.
● If these cytokeratin 18 are coated by epoxy, they will no
longer be recognized by the antibodies resulting in a
FALSE NEGATIVE result.

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