Sudan Black B staining (SBB staining) was performed using a previously published protocol (Georgakopoulou et al., 2013 (link)). In short, OCT-embedded and snap-frozen tissues were cryosectioned at a thickness of 10 μm with a Leica CM3050S cryomicrotome. Frozen sections were taken out of the refrigerator at −80°C, placed at RT for a few minutes and fixed in 1% (w/v) formaldehyde/PBS for 5 min at RT, and then rinsed gently with distilled water three times, incubated in 50% ethanol and 70% ethanol for 5 min each in turn. Next, frozen sections were stained in Sudan Black B solution (0.7 g SBB in 100 mL 70% ethanol) for 5 min at RT, and then rinsed quickly in 75% ethanol to remove excess staining solution, rinsed with distilled water three times. Finally, sections were left to stain in nuclear fast red solution for 3 min, washed in distilled water and mounted with glycerol. Images were taken with Olympus CKX41 microscope imaging system, and Image Pro Plus was used to quantify the Sudan Black B positive area.
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