Mouse zo 1

After enucleation, eye cups and retina were carefully removed. RPE flatmounts were fixed in 2% paraformaldehyde, stained with rabbit polyclonal antibodies against mouse ZO-1 (1:100; Invitrogen, Carlsbad, CA, USA), and visualized with Invitrogen Alexa Fluor 594 (Thermo Fisher Scientific, Waltham, MA, USA). For immunofluorescence staining for Cre expression, fresh, unfixed mouse eyes were embedded in optimal cutting temperature compound (Thermo Fisher Scientific), frozen in isopentane precooled by liquid nitrogen, and then cryosectioned. Using a Nikon A1R confocal microscope system (Nikon, Tokyo, Japan), flatmounts were imaged and assessed by a blinded operator. Degeneration in RPE flatmounts was defined using an established protocol.²⁰ (link)

Proteins were resolved on 10% SDS–PAGE gels, transferred to nitrocellulose membranes, blocked with 5% nonfat dry milk in TBS/0.1% Tween20 (TBS-T), washed three times in TBS-T, and incubated overnight in primary antibodies in 5% BSA/TBS (BSA cat. no. A7906; Sigma Aldrich) at 4°C. Antibody dilutions were as follows: rabbit Cx43 (Cell Signaling), 1:2000; mouse ZO-1 (Invitrogen), 1:1000; mouse clathrin heavy chain (BD Transduction), 1:2000; α-tubulin (cat. no. T2096, Sigma-Aldrich), 1:5000. Blots were washed three times in TBS-T and incubated in the appropriate secondary antibodies (goat anti-mouse-HRP and goat anti-rabbit HRP, cat. nos. G21040 and G21234, respectively; Invitrogen) for 30 min at 1:3000 dilution at RT. Relevant proteins were detected with enhanced chemiluminescent reagent. ImageJ (Nationl Institutes of Health) was used to quantify protein band intensities.

Cells were fixed with 4% formaldehyde in PBS at room temperature for 20 min, rinsed three times in PBS, permeabilised for 5 min with PBS containing 0.5% Triton X-100, and blocked in AbDil (PBS 5% BSA). Coverslips were then incubated in primary antibody (in PBS 1% BSA) overnight at 4°C, washed three times with PBS, and incubated in secondary fluorescently-conjugated antibodies. Antibodies used at 1:200 unless otherwise specified: rabbit Piezo1 (Novus, NBP1–78446); mouse S1P (Santa Cruz, CA sc-48356); rabbit KCNA1 (Alomone Labs, APC-161); rabbit KCNA2 (Alomone Labs, APC-010); rabbit LRRC8A (Alomone labs, AAC-001); mouse ZO1 (Invitrogen, 33–9100); rabbit ENaC antibodies SCNNA1 (Invitrogen, PA1–920A), SCNNB1(Invitrogen, PA5–28909), and SCNNG1(Invitrogen PA5–77797). Alexa Fluor 488, 568 and 647 goat anti–mouse and anti–rabbit IgG were used as secondary antibodies (Invitrogen). F-actin was stained using either conjugated 488 or 568 Phalloidin (66μM) at 1:500 and DNA was detected with 1μg/ml DAPI (Themofisher) in all fixed cell experiments.