Anti dlk

Eyes were harvested and fixed for 1 h in 4% PFA. Following cryopreservation in 10%, 20%, and 30% sucrose at 4°C for 30 min, 2 h and overnight, respectively, eyes were embedded in Tissue-Tek OCT compound (Electron Microscopy Sciences) and sectioned at 10 μm using a cryostat. The following primary antibodies were incubated with tissue sections overnight at 4°C: anti-Iba1 (1:200, Abcam), anti-CD68 (1:250, Bio-Rad), anti-GFAP (1:500, Abcam), anti-DLK (1:100, Genetex), anti-Atf3 (1:200, Novus Biologicals), anti-GFP (1:500, Abcam), anti-Il22Ra1 (1:200, Bioss), anti-Rbpms (1:100; PhosphoSolutions), anti-βIII-tubulin antibody (1:500, Promega). Following primary antibody application and washing, tissue was subsequently incubated in AF594, AF546, AF647, or AF488 secondary antibodies (1:400; Invitrogen, Thermo Fisher) for 1 h. DAPI (1:1000; Invitrogen) was used to label nuclei. Images were captured using an Axio Imager M2 (Zeiss), a LSM 880 confocal (Zeiss), or a TCS SP8 confocal (Leica). To visualize AAV infection in wholemount retinas, tissue was stained with an anti-GFP (1:500, Abcam) antibody for 3 d at 4°C. Retinas were subsequently washed in PBS, incubated with an AF488 secondary antibody (1:400; Invitrogen) for 2 h at room temperature, and washed in PBS.