Ha720050

Six-micrometer-thick paraformaldehyde-fixed, paraffin-embedded sections of diaphragm were deparaffinized for immunofluorescent staining. All sections were incubated with anti-collagen III primary antibody (HA720050 1:500 HuaAn Biotechnology, China) at 4 °C overnight. Tissue was then incubated with secondary antibodies against rabbit (A-11035, 1:500, Invitrogen, United States) labeled with Alexa Fluor 546 at 37 °C for 30 min. Then, all slides were stained with Hoechst (H1399, 1:500, Invitrogen, United States) and wheat germ agglutinin (WGA) (W11261, 1:1000, Invitrogen, United States) for 10 min 37 °C. Finally, the slides were observed under a confocal microscope (Olympus IX83-FV3000-OSR, Tokyo, Japan), and representative views were selected and photographed.

The diaphragm protein lysates were subjected to SDS‒PAGE gels for electrophoresis for 90 min and then transferred onto a polyvinylidene fluoride membrane (IPVH00010, Millipore, United States). The membrane was blocked and then incubated with primary antibodies against TGF-β1 (HA500496 1:1000, HuaAn Biotechnology, China), α-SMA (ER1003 1:1000, HuaAn Biotechnology, China), COL3 (HA720050 1:1000, HuaAn Biotechnology, China), COL1A1 (ET1609-68, 1:1000, HuaAn Biotechnology, China), COL6A1 (A9236, 1:1000, ABclonal, Woburn, MA, USA), and COL6A2 (A3796, 1:1000, ABclonal, Woburn, MA, USA) overnight at 4 ℃ and secondary antibodies at room temperature for 90 min and then incubated with ECL reagent. The final reported data of COL1A1, COL3, COL6A1, COL6A2, TGF-β1 and α-SMA were the band densities normalized to that of GAPDH.