Aqua poly mount reagent

All immunofluorescence staining protocols are previously published [19 (link)]. Briefly, sections were permeabilized with PBS containing 0.4% of Triton X-100 then blocked with PBS containing 5% normal donkey serum and 0.1% Triton X-100 for 1 h at room temperature and incubated with primary antibody of interest overnight at 4 °C. The primary antibodies that were used are shown in Table 1. The sections were then incubated for 1 h at room temperature with species-specific secondary antibodies directly conjugated to Alexa fluorophores (1:1000, Invitrogen) followed by nuclei staining with Hoechst. A coverslip was mounted onto sections using aqua poly/mount reagent (Polysciences, Warrington, PA, USA). Images were captured using a Zeiss Axio Observer Z1 epifluorescence microscope and Axiovision software with the appropriate excitation and emission filters.
For vertical retina Tuj quantification, the IPLs of six equidistant regions of interest (ROI) per retina were analyzed using ImageJ software. For optic nerve, three cross sectional regions were quantified as previously described [19 (link)]. ImageJ software was used to quantify mean fluorescent intensity (MFI) of the immunofluorescent signal by persons blinded to sample information. For enumerating glia and T cells, Zen Blue software (Carl Zeiss, Oberkochen, Germany) was used.