M1005 2

Paraffin sections of 3 µm thick were prepared and stained according to the standard operating instructions of the Immunohistochemistry Kit (SA1027, Boster, Wuhan, China). The histological sections were dewaxed and hydrated, followed by antigen repairing for 20 minutes. The sections incubated with 3% hydrogen peroxide–methanol solution protected from light at 37°C for 15 minutes. The primary antibody used was Piezo1 (1:50, M1005-2; Huabio, Hangzhou, China) was then added overnight at 4°C. Anti-mouse secondary antibody (SA1027, Boster) was incubated at room temperature for 1 hour. Immunoreactivity was subsequently tested with the DAB reagent (ZSGB-BIO). Then the sections were restained with hematoxylin and dehydrated transparent. Finally, the sections were sealed with neutral resin and observed under a light microscope (Leica).

RIPA lysis buffer (Solarbio) was added to the retina and well cut up with scissors, followed by further lysis with ultrasound (Qsonica, Newtown, CT, USA). The supernatant was collected after centrifugation at 12,000×g for 15 minutes, and protein concentration was determined by the BCA kit (Beyotime). The samples were mixed with a 5× protein loading buffer (Beyotime) and then placed at 100°C for 15 minutes; the protein samples were loaded on 8% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. Then, the membranes were blocked with 5% nonfat milk at room temperature for 1.5 hours and incubated with the primary antibody (anti-Piezo1, 1:500, M1005-2, Huabio; anti-GAPDH, 1:5000, ET1601-4, Huabio) at 4°C overnight. After that, the membranes were incubated with a secondary antibody (1:10,000; Boster) for 1 hour at room temperature. Finally, the bands were exposed using the ECL developer solution (Biosharp), and the optical density was quantified using ImageJ software.