Vectashield hardset antifade mounting media

For imaging studies, HTLA cells grown on poly-l-lysine-coated coverslips were fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature. Cells were then washed three times with PBS and incubated with the Anti-FLAG M2 monoclonal antibody (Sigma-Aldrich Cat# F1804, [RRID:AB_262044], Sigma, St Louis, MO) at room temperature for 1 h (1: 500 dilution) as previously described. After the 1 h incubation, coverslips were transferred to 4°C and left to incubate further overnight. After washing, the Alexa Fluor 488 (anti-mouse) (Molecular Probes Cat# A-11029, [RRID:AB_138404], Life Technologies, Thermo Fisher Scientific, Grand Island, NY) secondary antibody was used as required for 1 h. HTLA cells were washed three times with PBS followed by mounting the coverslips onto slides using Vectashield Hardset Antifade Mounting Media (Vector Laboratories Cat# H-1400, [RRID:AB_2336787], Vector Laboratories Inc., Burlingame, CA). Images were acquired using the Leica PAULA Smart Cell Imager (Leica Microsystems, Buffalo Grove, IL).

Mice were sacrificed using CO₂ inhalation and perfused transcardially (exsanguinated) with 15 mL 0.01 M phosphate-buffered saline (PBS) followed by 20 mL of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. After 24 h post-fix in 4% PFA, brains were cryoprotected in successive incubations of 15 and 30% sucrose in PBS over 3 d. Frozen tissue was cut coronally on a sliding cryostat (Leica Microsystems, Germany) at 40 μm, mounted on gelatinized slides and stored at −80 °C until further use. Subsequently, sections were air-dried at RT for 20 min before being washed extensively in PBS followed by a permeabilization/block step in 4% normal goat serum, 1% bovine serum albumin and 0.3% Triton-X in PBS. Sections were then incubated with a mouse monoclonal anti-GFAP primary antibody conjugated with Cy-3 (1:750; C9205 Sigma Millipore) for 24 h at 4 °C in a humidified chamber with thermoplastic (Parafilm M) coverslips to prevent drying. After washing with PBS, sections were coverslipped with Vectashield Hardset antifade mounting media containing DAPI counterstain (Vector Laboratories, Burlingame, CA).

Retinal sections were stained using the in situ cell death Detection kit, TMR red (12156792910, Roche Applied Sciences) according to the manufacturer’s instructions. Briefly, retinal sections were washed three times for 5 minutes in PBS and permeabilized for 2 minutes in freshly prepared cold 0.1% Triton X-100 and 0.1% sodium citrate solution. The slides were then washed twice with PBS before being resuspended in the TUNEL reaction mixture for 1 hour in a humidified chamber at 37°C. Slides were washed with PBS before continuing with the immunohistochemistry staining protocol. Nuclei were counterstained with DAPI (1:5000) and cover slipped using Vectashield hardset antifade mounting media (Vector Labs). Slides were imaged using the BZ-X700 microscope (Keyence).