Figure 2. Validation of ISH-PLA using SMC lineage tracing mouse model.
(a) SMC lineage tracing was done by crossing Myh11-CreERT2 transgenic mice with ROSA26 STOPfloxeYFP+/+ mice and treating mice with tamoxifen between six and eight weeks of age thereby providing SMC-specific and permanent lineage tagging of SMCswith eYFP(n =5).(b) Immunostaining of the aorta of SMC-eYFP+/+ mice for eYFP and ACTA2 with (bottom image) and without (top image) tamoxifen injections. eYFP expression was observed only after tamoxifen treatmentand was exclusively observed in SMCs within the media (M), compared with the intima (I) and the adventitia (A) negative for eYFP staining. L: lumen. Scale bar = 10 μm. (c) Assessment of eYFP expression in heart tissue sections of SMC-eYFP+/+ mice by immunostaining for eYFP (cyan), ACTA2 (red) and Dapi (blue). eYFP expression is strictly restricted to ACTA2+ cells. (d) SMC-eYFP−/−mice present a complete lack of eYFP expression in ACTA2+ cells in heart tissue sections. Scale bar (c–d) = 100 μm. e. Results of ISH-PLA in aortas from SMC-eYFP+/+ mice showing that Myh11 H3K4dime PLA positivity was restricted to eYFP+ medial SMCs(arrows)(media: M). No Myh11 H3K4dime PLA signal was detected in ECs (stars) (intima: I). L: lumen. i–iii show eYFP+ PLA+ SMCs at higher magnification with i) eYFP, ii) PLA and iii) merge. Scale bar = 10 μm. f. Negative control wherein ISH was done using an empty biotinylated vector in SMC-eYFP+/+ mice. No Myh11 H3K4dime PLA+ cells were identified.Scale bar = 10 μm.