Eyes were freshly dissected after transcardial perfusion with 0.1M PBS, immersion fixed in 4% PFA for 15 to 30 minutes, then whole-mount retinas were dissected for RGC labeling with RNA binding protein with multiple splicing (RBPMS), as previously described.
30 ONs attached to the brain were dissected from the skull, then post fixed for 24 hours in 4% PFA. The following antibodies were used: RBPMS (an RGC marker, 1:200, GTX118619; GeneTex, Irvine, CA, USA), glial fibrillary acidic protein (GFAP, 1:500, ab53554; Abcam, Cambridge, MA, USA), ionized calcium binding adaptor molecule 1 (Iba-1, a microglia marker, 1:200, 019-10741; Wako, Richmond, VA, USA), neurofilament 200 (NF200, 1:100, N4142; Sigma-Aldrich), p62 (sequestosome 1 [SQSTM1], 1:100, ab56416; Abcam), HIF-1α (1:100, sc-13515; Santa Cruz Biotechnology, Dallas, TX, USA), HIF-2α (1:100, sc-13596; Santa Cruz Biotechnology), and superoxide dismutase 2 (SOD2, 1:100, sc-133134; Santa Cruz Biotechnology). The above dilutions of primary antibodies were determined by optimization with different serial dilutions. Whole mounts and sections were rinsed in PBS, blocked with 5% DKS, and 1% Triton X-100 in 1X PBS, quenched using 0.3% H
2O
2, then incubated with primary antibodies prepared in PBS-T-BSA for 2 days (whole mounts) or overnight (sections) at 4°C. Tissue and sections were rinsed in PBS then blocked in PBS-T-BSA. Secondary antibodies (1:250; Alexa- Fluor488, 594, or 647; Jackson ImmunoResearch) prepared in PBS-T-BSA were added and incubated overnight at 4°C (whole mount) or for 2 hours (sections). DAPI (4′,6-diamidino-2-phenylindole; 1:2000) was applied, then rinsed before sections were cover-slipped using Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Six to 10 representative slides (four sections/slide) were imaged. To confirm specificity, we incubated sections with secondary antibodies but without primary antibodies. Whole mounts were mounted nerve fiber layer (NFL) side up on slides, then cover-slipped. Images were captured using a Leica DMi8 confocal microscope integrated with Leica confocal microscope (mentioned above) or Olympus FV-1000 confocal microscope (Olympus, Cedar Valley, PA, USA). Mean intensity of p62 and SOD2 in the regions of interest were analyzed using four sections per slide, and three slides per retina or ON using Image J.