Nbp1 78624

Mice were anesthetized with ketamine (200 mg/ml) and xylacine (50 mg/ml), and they were perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). The brain was then removed from each mouse and after post-fixing by immersion in 4% PFA (24 h, 4 °C), coronal vibratome sections (50 μm; VT 1000S, Leica) were obtained and then incubated for 30 min in blocking solution at room temperature (3% goat serum in TTBS). The sections were immunostained overnight at 4 °C with the primary antibodies and after washing three times in PBS (5 min) the secondary antibodies were applied for 2 h at room temperature. After three 5 min rinses with PBS, the nuclei were stained with DAPI (2 μM; Life Technology, Carlsbad, USA). The antibodies used were: rabbit anti-Piezo2 (1:100; NBP1-78624, Novus Biological, Littleton, USA); chicken anti-GFP (1:1000; AB13970, Abcam, Cambridge, UK), goat-anti rabbit AlexaFluor-594 (1:1000; A11012) and goat-anti chicken AlexaFluor-488 (1:2000; A11039, Life Technology).

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).