INFILTRATION (impregnation)

- tissue are placed in a substance that will completely fill all cavities and interstices
~~ if holes di ma fill in kay mag cause ug distortion or damage
~~ fill in ang mga holes sa cavities pra maganda pag cut di dali mabuak?
- Gives firm consistency to tissue
- Facilitate handling & cutting
Types of Tissue Impregnation
PARAFFIN WAX
- Polycrystaline mixture of solid hydrocarbons
- 56oC
- More blocks are processed in a short time
- Serial sections are easily obtained
- Routine & special staining can be easily done
~~ four changes paraffin wax to ensure that the clearing agent is completely removed

Celloidin (Collodion)
- permits cutting of thick tissue sections; or spxs with large cavities/ hollow spaces w.c
tend to collapse
A. WET (Ether + 70% alcohol); for bones teeth, large brain sections
B. DRY (Gilson’s = components: chloroform and Cedarwood oil = clearing agents); for
whole eye sections
~~ to make tissue transparent

Gelatin
- used when dehydration and clearing are avoided
- For histochem and enzyme studies
- For FROZEN sections
- Main source,
- Organic in nature, thus easier na ma-tubuan ng molds
> to avoid growth of molds
Add 4% phenol on the gelatin to prevent growth of molds during infiltration
Plastic (Resin)
- Used widely for light and electron microscopy
Nitrocellulose Method

MODIFIED PARAFFIN WAXES

- Better properties and additional substances to improve ribboning
- To improve adhesion between wax and tissue
~add phenanthrene

● Stearic acid (increase hardness)

● Phenanthrene or spermaceti (decrease MP melting point)
● Ceresin, beeswax (improve adhesion)
 ● Piccolyte 115 (thermolastic terpene resin)
● Plastic polymers (ex. Polyethylene wa
● Dimethyl sulfoxide (DMSO)
~~ DMSO = reduce infiltration time as well as cause product thin sections during
microtomy; alter permeability of tissues para dali ma remove ang water
~~ DMSO:
+ Decrease infiltration time
+ can create thin sections
+ alter tissue permeability by removing water
>>>> Substitute
A. Paraplast
B. Embedol
C. Bioloid
D. Tissue Mat
E. Ester wax: can be used w/o clearing
F. Carbowax: used w/o dehydration or if clearing is not needed

● Aqueous Media
- Agar
- Gelatine
- Sodium carboxymethyl cellulose
- Polyvinyl alcohol
+ Highly polar, water soluble
+ Histochemical studies of lipids and enzymes

● Water MiscibleMedia
- PEGs polyethylene glycose
+ heat and solvent abile lipids & proteins (madaling masira)
+ Prevents tse shrinkage & damage
+ Less elastic, denser

EMBEDDING
- AKA casting or blocking
- Process by w/c tissues are precisely arranged in a mold surrounded by a medium such
as agar, gelatine, or wax which when solidified will provide sufficient external support
during sectioning
- Infiltrating medium are same as embedding medium
—— Immerse infiltrated tse in melted paraffin wax to then wait na mag harden

~~ Requirements for embedding are as follows

+ a supply of clean, filtered paraffin wax held at 2-4C above its melting point (embedding
medium dapat di solidified)
+ A cold plate to rapidly cool the wax (para mag solidify)
+ A supply of molds in which to embed the tissues.
 - Paper boat? Ahhahaha basta common nga embedding ginagamit
- cold plate: to facilitate cooling; can occupy 60-60 blocks
Small surface are of paraffin wax = l

- Has paraffin reservoir wherein crystals are placed para mag melt
~~ Temp is adjustable (should be greater than the melting point)

NOTE:
- Melting point of wax should be around 45-60 oC
~ Once in room temperature lab temp: (20-25 or 25oC), temperature of parrin wax should be
held around 54-58oC
~ If lab temp is 15 to 18oC or lower, paraffin should be in constant 50-54oC
~Paraffin oven or incubator should be maintained 2-5 oC above the melting point of wax of the
paraffin wax hahahahahah yawa ambot pataka ra

Embedding procedure
 + Mold should not be congested; it should have more spaces

+ Usual paper mold used: Paper boat

+ place label sa mold itself, pero there are instances na sa paper??

Alternative Embedding Media

Plastic (Resins)
Needed when:
- Heat or reagent-labile tissues
- Hard or dense tissues
- Tissues with poor adhesion with wax are processed
- Very thick/thin sections are needed
- Sectioning whole organs
+ epoxy: Has vinylcyclohexane dioxide, carcinogenic; thus not usually used
~~ Benzoyl Peroxide: Added to plastic embedding medium; act as a catalyst polymerization to
hasten that can decompose
+ Polyester
+ Acrylic
Resin Removal
- Render sa staining clarity maong dapat I remove
- hard resins provide a barrier to dyes
- Markedly reduce staining clarity
- Must be done before staining
- Epoxy resins are degraded by reagents such as;
+ Alcoholic NaOH
+ KOH
+ Na methoxide
+ Bromine vapor
Double Embedding
- Combination of celloidin & paraffin embedding
~ 2% celloidin for around 3 days then transfer sa paraffin wax
- Used when tissues require external support or particular pre-embedment orientation
- Organs benefited: bone, brain, muscle, large pcs of dense
Celluloses Nitrate/ Low viscosity Nitrocellulose
- Considerably less shrinkage
- Improved cutting qualities of large blocks of dense tissue
- Ex: tooth, embryos, facilitate production of sections of brain (preserving relationships of
all tissue layers of different consistency)
- Disadvantage:
- When dry, dropping the container of dry LVN can cause explosion
~~ LVN: Use of oleum ricini and castor oil may cause the tissue to crack
> add plasticizers to LVN during embedding to avoid tissue crack

Factors affecting paraffin wax impregnation

1. Nature and size of the tissues

2. Clearing agent used prior to impregnation

Example:
+ Benzene and Xylene: easily removed from tissues
+ Chloroform and Cedarwood Oil: difficult to remove

PRECAUTIONS OBSERVED IN PARAFFIN WAX IMPREGNATION

1. Avoid prolonged treatment of tissues in melted paraffin.
2. Avoid infiltration in overheated paraffin (above 60oC)
3. Oven must be maintained @2-5oC above the M.P. (melting point) of wax
4. Wax must be pure, free from dust, foreign matter
5. Fresh wax should be filtered before use in a wax oven
6. Paraffin wax should be used only twice
7. Hard wax with higher MP requires a heavy-duty type of microtome
8. Hard and dense tissues require wax with higher M.P. than soft ones

ORIENTATION
- Process by w/c tissue is arranged in the mold during embedding, on the microtome
before cutting and on the slide before staining
 - Encompasses;
+ embedding
+ Microtomy
+ staining
—— Tissues are blocked with the surface to be cut facing down in a mold in diagonal position
para tanan makita sa surface sa tissue

Practical points to consider

1. Elongated tissues; placed diagonally across the block
2. Tubular and walled specimens (cysts, fallopian tubes, GIT tse, vas deferens); en face to
provide cross sections showing all tissue layers
* en face: in front tse block;
3. Epithelial surface (skin): section in a plane at a right angle to the surface (hairy or
keratinized epithelia are oriented to face the knife diagonally)
4. Multiple tissue pieces: aligned across the long axis and the center of the mold; not
placed randomly

- Cervix: cut first sa dense part to avoid?? Patay lods wako ka kuan ani huhu
- Skin: epidermis should be the last part na i-cut

● Orient biopsy specimen submitted EN TOTO ON END or ON EDGE

● USE CONSISTENCY AND ALIGNING
 ● Orient biopsy specimens submitted BISECTED (kaisa ra gi cut) or DISSECTED (multiple
gi cut) on the CUT EDGE
● Orient “breadloaf” dissected specimens with the largest cut edge down
● When possible, arrange multiple sample pieces in the block to permit maximum
representation on the resulting microslide
~~ Note: prefers to have a pair for each ribbon on the slide. Max of 8 total sections on 1 slide
● Orient needle core pieces:
1. Without overlapping or layering
2. Arrange to permit maximum representation on the slide
~~ Ideal: 3-4 per slide; from longest down to the shortest
~~ No overlapping; not arrange or tamped

● Cut and orient VAS vas difference, FTS fallopian tube, and TAR lumen down

Factors affecting Tissue Processing

Properties of the Tissue
● Fixation
● Solvent Effects
- dissolution of lipid-rich structures = increased porosity = increased shrinkage
● Tissue Density
- Affects infiltration & microtomy
● Tissue Modifiers
- Phenol: swells collagen /elastic fibers
- Enhances polymerization & tse. Porosity
● Tissue Porosity
- Natural & art;factual pores
● Concentration
- increased in graded increments
● Evaporation rate
- Increased ER = decreased contamination
● Density (for embedding medium)
- must match the densest tse component to avoid deformation
● Elasticity & Plasticity (embedding medium)
- to recover from deformation
- To facilitate thin sectioning
● Polarity
- Changed gradually (from highly polar fixative & dehydrant to non-polar embedding
medium)
● Viscosity
Processing Condition
● Agitation
+ Manual: shaking, rotors, magnetic stirrers
+ Automated: rotary/vertical motion, tidal action of fluids
+ Ultrasonic stimulated: high freq. agitation
● Specimen-fluid volume ratio
● Heat
- Hasten rxn
● Vacuum
- Faster penetration
~ Recommended dehydrating agent to revive dessicated:
- 50 ml Water & 30 ml absolute alcohol + 20 ml of 5% aqueous sol’n of Sodium
bicarbonate (Na2Co3)
~ Formulation of Anderson-Gordon rgt:
- 70 ml of 70% ethanol + 30 ml glycerol + 1 g dithionite
> Procedure:
— Soak tissue for several hours,usually overnight then process from the dehydration stage in
the usual manner
~ Sandinson’s rgt
- 30 ml 96% ethanol + 50 ml of 1% aq formalin + 20 ml of 5% aq Sodium carbonate
(NaCo3)
> Procedure:
— Soak tissues for 12 to 18 hours, then process from the dehydration stage in the usual
manner