Fluoromount g mounting media

Embryos were fixed in 4% paraformaldehyde for 2–24 hours depending on the age, washed briefly with PBS, and transferred to a solution of 30% sucrose in PBS until they sank. Embryos were then embedded in Neg-50 (Richard Allan Scientific, San Diego, CA), frozen on dry ice, and stored at −80°C until they were sectioned. Immunohistochemistry was done using 20-um frozen sections cut on a Leica CM3050 S cryostat (Buffalo Grove, IL, USA). Prior to staining, sections were equilibrated to room temperature for at least 30 minutes. Antigen retrieval was performed using a citrate-based buffer (Antigen Unmasking Solution H-3300; Vector Laboratories, Burlingame, CA, USA) and steam for 20 minutes. Sections were then blocked in 2% normal goat serum in PBS with 0.1% Triton X-100 (PBS-TritonX), incubated in primary antibody overnight at 4°C, washed 3 × 3 minutes with PBS-TritonX, incubated in secondary antibody for 1 hour at room temperature, washed 2 × 3 minutes with PBS-TritonX, stained with DAPI (1:10,000) for 3’ in PBS, washed one last time for 3 minutes with PBS, and mounted in Fluoromount-G mounting media (Thermo Fisher Scientific, Waltham, MA, USA). Images were captured on a Zeiss LSM 700 series laser scanning confocal microscope (Carl Zeiss Meditec, Oberkochen, Germany).

OTC embedded ears from mice were sectioned and stained for basement membrane IgG deposition using an anti-mouse IgG-FITC conjugated Ab (Invitrogen, no. 62-6511, 1:200 dilution in 1% BSA/PBS). Slides were mounted using Fluoromount-G mounting media (Thermo Fisher Scientific, no. 00-4958-02) with DAPI (Thermo Fisher Scientific, no. 00-4959-52). Slides were imaged using a Leica SP8 confocal microscope.

OTC embedded ears from mice were sectioned and stained for basement membrane IgG deposition using an anti-mouse IgG-FITC conjugated Ab (Invitrogen, no. 62-6511, 1:200 dilution in 1% BSA/PBS). Slides were mounted using Fluoromount-G mounting media (Thermo Fisher Scientific, no. 00-4958-02) with DAPI (Thermo Fisher Scientific, no. 00-4959-52). Slides were imaged using a Leica SP8 confocal microscope.

BVOs were rinsed twice in PBS and then fixed in 4% paraformaldehyde (PFA) for 1 h at room temperature (RT) and washed twice in PBS. BVOs were dehydrated in 20% sucrose at 4 °C overnight, then embedded in Gelatin solution and stored at −20 °C for cryosectioning. The frozen BVOs were sectioned using NX70 Cryostat (Thermo Scientific) to 20 and/or 70-μm thickness. Frozen sections were then washed in PBS for 5 min and then permeabilized and blocked in Blocking buffer for 2 h at RT. Primary antibodies were diluted in Blocking buffer and incubated overnight in a cold room. Antibodies used and dilution factors’ information are available in Supplementary Table S1. On the following day, sections were washed three times in PBS with 0.1% TritonX-100 (PBS-T) and then incubated with labelled secondary antibodies for 2 h at RT. After two washes in PBS-T, the sections were counterstained with DAPI and mounted with Fluoromount-G mounting media (Thermo, 00-4958-02).
For immunostaining intact BVOs and VNs were rinsed in PBS then fixed in 4% PFA for 30 min at RT and washed twice in PBS. The fixed VNs were stored in PBS at 4 °C for up to a month. Stained BVOs were mounted into iSpacer (0.5 mm deep imaging Spacer, SunJin Lab). Samples were viewed and imaged using the Spinning Disk Confocal System (Nikon) and the Operetta CLS High-Content Analysis System (PerkinElmer) at a 20x or 40x magnification.

Electroporated retinas were harvested at P12 (P20 for rd1 mice). Retinas were fixed in 4% formaldehyde for 30 min, transferred to phosphate-buffered saline (PBS) for 10 min, and transferred to 15% sucrose in PBS for 30 min, all at room temperature. Retinas were then embedded in OCT, flash frozen, and stored at −80°C. 12 µm sections were made using a cryostat, placed on glass slides, and mounted with Fluoromount-G mounting media (Thermo Fisher, 00-4958-02). Retinas from rd1 mice were stained for GFAP using chicken anti GFAP primary antibody (1:3000, Aves Labs, cat.#: GFAP) and secondary antibody conjugated to AlexaFluor 488 (Jackson ImmunoResearch). Images were taken using a Nikon Ti2 inverted microscope (spinning disk confocal) with a ×40 oil immersion objective lens.