Retinas were harvested after the animals were killed by pentobarbitone overdose. Retinas were homogenized in RIPA buffer that contained 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 10 mM sodium pyrophosphate, 1.0 mM Na3VO4, 1.0 mM PMSF, and complete protease inhibitor cocktail (Roche). The protein concentration in each sample was determined by a BCA protein assay (Bio-Rad Laboratories). Total protein in the samples was electrophoresed on 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Primary antibodies were incubated overnight on a rocker at 4℃, followed by an appropriate secondary antibody conjugated to horseradish peroxidase (HRP) Signals were visualized using ECL-Plus Western blotting detection reagents (Thermo Fisher Scientific 1,863,096 and 1,863,097). All experiments were repeated three times.
Primary antibodies used were rabbit anti-GFAP (1:1000, 16,825–1-AP, Proteintech, subsidiary in Wuhan, China); anti-EAAT4 (1:3000, EAAT41-A, Alpha Diagnostic international, USA); anti-ID1 (1:500,18,475–1-AP, Proteintech, subsidiary in Wuhan, China); anti-EDNRB (1:500, 20,964–1-AP, Proteintech, subsidiary in Wuhan, China); anti-BMP6 (1:1000, A4538, ABclonal, China); anti-PIEZO2 (1:500, NBP1-78,624, Novus Biologicals, Littleton, CO, USA); anti-GAP (1:5000, 5174S, Cell Signaling Technology, USA).
Primary antibodies used were rabbit anti-GFAP (1:1000, 16,825–1-AP, Proteintech, subsidiary in Wuhan, China); anti-EAAT4 (1:3000, EAAT41-A, Alpha Diagnostic international, USA); anti-ID1 (1:500,18,475–1-AP, Proteintech, subsidiary in Wuhan, China); anti-EDNRB (1:500, 20,964–1-AP, Proteintech, subsidiary in Wuhan, China); anti-BMP6 (1:1000, A4538, ABclonal, China); anti-PIEZO2 (1:500, NBP1-78,624, Novus Biologicals, Littleton, CO, USA); anti-GAP (1:5000, 5174S, Cell Signaling Technology, USA).