Each tissue sample was fixed with 4% paraformaldehyde, paraffin-embedded, and sectioned into 3 μm-thick sections. For hematoxylin and eosin (H&E) staining, the slides were deparaffinized, rehydrated, and stained with H&E. Masson’s trichrome staining (connective tissue stain) was performed according to the manufacturer’s instructions (#SS1026-MAB-500, CANCER). Briefly, cryosection slides were placed in preheated Bouin’s fluid for 60 min, followed by a 10 min cooling period. The slides were rinsed in tap water until the sections were completely clear and then washed once in distilled water. The slides were then stained with equal volumes of Weigert’s A and B for 5 min and rinsed with running tap water for 2 min. Next, the slides were exposed to Biebrich scarlet-acid fuchsin solution for 15 min and rinsed with distilled water. The slides were differentiated in phosphomolybdic/phosphotungstic acid solution until collagen was no longer red and then rinsed with distilled water. Without further rinsing, the slides were treated with aniline blue solution for 5–10 min, followed by treatment with 1% acetic acid for 3–5 min and rapid dehydration with two changes of 95% and 100% ethanol. Finally, the slides were incubated with xylene and mounted with balsam.
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