2.4.3. Immunofluorescent staining for collagen type I and collagen type II

To identify the absence or presence of the ECM components collagen type I and collagen type II, we performed immunofluorescent analysis. 5 μm sections were deparaffinized after treatment with UltraClear™ solution followed by ethanol and distilled water. Due to the formation of methylene bridges during fixation, the slides were incubated for 30 min at room temperature in an antigen retrieval enzyme, protease XXV (AP-9006–005, Thermo Scientific, Massachusetts, US). To increase the specificity of the antibodies, the slides were incubated in hyaluronidase (H6254, Sigma-Aldrich, Missouri, US) for 30 min at 37 °C. The sections were then incubated in bovine serum albumin (BSA) 5% w/v to reduce non-specific binding of the antibodies. After BSA incubation, the pellet slices were incubated in primary antibodies: rabbit anti-collagen I (CL50111AP-1, Cedarlane, Ontario, Canada) and mouse anti-collagen II (II-II6B3, Developmental Studies Hybridoma Bank, Iowa, US) overnight at 4 °C with a 1:200 dilution. The preceding step was followed by incubation with a fluorescently-conjugated secondary antibody at a dilution of 1:200 with goat anti-rabbit IgG (H&L Alexa Fluor 594, Abcam, UK) for collagen type I and goat anti-mouse IgG (H&L Alexa Fluor 488, Abcam, UK) for collagen type II. The sections were stained with DAPI (4′, 6-diamidino-2-phenylindole, Cedarlane) to stain the cell nuclei, and mounted with a 1:1 glycerol-PBS solution. Immunofluorescent images were visualized by an Eclipse Ti-S microscope (Nikon Canada, Ontario, Canada).