Anti β 3 tubulin

Cultures were fixed in 4% paraformaldehyde on DIV 14 for 1 h. Cell permeabilization was done with Triton X-100 (Sigma-Aldrich) in PBS for 2 h on a shaking platform. Cultures were then blocked using 10% goat serum in PBS for 1 h. Antibodies (Anti-GFAP antibody (Abcam, ab7260) conjugated to Alexa Fluor 647(Thermo Fisher, A27040) and Anti-NeuN (Millipore Sigma, MAB377) or Anti-β-3-Tubulin (Thermo Fisher Scientific, MA1-118) conjugated to Alexa Fluor 488 (Thermo Fisher Scientific, A28175) were then applied to the cultures on a shaker at +4 °C for 48 h. Cultures were counterstained with DAPI. A drop of Fluoro-Gel (Electron Microscopy Sciences, Catalog # 17985-10) was added on the fixed and stained culture. Spacer glass coverslips were placed at two sides of the culture and another coverslip was added on top of the culture to create a sandwich. Cultures were then imaged using a confocal microscope (Zeiss LSM 510 META, Germany) with ×40 objective. Distance between optical slices was 1 μm, and cultures were imaged over their entire depth. Images were then processed in Fiji (ImageJ).

Retinal flat mounts were prepared, stained, and analyzed as described [32 (link)]. Primary antibodies included rat anti-CD11b, clone M1/70, BD Bioscience; rat anti-Ki67, clone SolA15, eBioscience; and anti-β3-tubulin, ThermoFisher. Secondary antibodies (Invitrogen) included Alexa Fluor 594 donkey anti-rat IgG; or biotinylated anti-rabbit IgG and Alexa Fluor 488/streptavidin; or biotinylated anti-rat IgG and Alexa Fluor 350/Streptavidin. Cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories). GFP and YFP were detected by their fluorescence. For cell quantification, 8 individual 0.19 mm² fields (4 central, 4 peripheral) per retina were examined. The total number of cells through the entire retina within a field or contained within the field of the indicated retinal cell layer was counted. Results expressed as a mean number of cells per field or total cells per retina which was calculated based on a retinal volume of 2.7 mm³.