Mouse anti apc

The following antibodies were used in this study: mouse anti-Galectin-3 (BioLegend, 126702), goat anti-human GPNMB (R&D Systems, AF2550), goat anti-mouse GPNMB (R&D Systems, AF2330), rat anti-mouse LAMP1 (BD Biosciences, 553792), rabbit anti-IBA-1 (Wako, 01919741), goat anti-AIF-1/Iba1 (Novus Biologicals, NB100–1028), rat anti-CD68 (Bio-Rad, MCA1957), and sheep anti-PGRN (R&D Systems, AF2557), mouse anti-MBP (Millipore, SMI-99), Goat anti-Olig2 (R&D Systems, AF2418), mouse anti-APC (Millipore, OP80), rabbit anti-Perilipin2 (Proteintech Group,15294-1-AP), sheep anti-TREM2 (R&D Systems, AF1729). Detailed information is provided in Supplementary Table 1.
The following reagents were also used in the study: Dulbecco’s modified Eagle’s medium (DMEM)(Cellgro, 10–017-CV), Hanks’ Balanced Salt Solution (HBSS) (Cellgro, 21–020-CV), Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM/F-12)(Cellgro, 10–092-CV), 0.25% Trypsin (Corning, 25–053-CI), Autofluorescence Quencher (Biotium, 23007), Odyssey blocking buffer (LI-COR Biosciences, 927–40000), and O.C.T compound (Electron Microscopy Sciences, 62550–01).

Animals were sacrificed following approved IACUC methods. For treatment (PLX5622 vs control diet) controls and determining effect of CCAO on the retina, the MONs or retinas were removed acutely and post-fixed for 24 hours in 4% paraformaldehyde (PFA). For IHC analysis of MONs that were exposed to OGD (with or without IPC), MONs were removed from the perfusion chamber at the conclusion of recordings and drop-fixed for 24 hours in 4% PFA. After fixation, MONs were rinsed in phosphate buffered saline (PBS), cryo-protected in 10%, 20%, and 30% sucrose, embedded in optimal cutting temperature (OCT) compound (Fisher), and sectioned at 12 μm on a cryostat. Slides were blocked in 10% donkey serum (Jackson ImmunoResearch) and 0.1% Triton X-100 (Sigma) for 30 min. Primary antibodies used: goat anti-Iba1 (Abcam, 1:500) or rabbit anti-Iba1 (Wako, 1:250), rabbit anti-TMEM119 (Abcam, 1:1000), sheep anti-Chx10/Vsx2 (Exalpha, 1:300), mouse anti-HuC/D (Invitrogen, 1:500), mouse anti-SMI31 (BioLegend, 1:500), and mouse anti-APC (Millipore, 1:200). Slides were incubated with appropriate secondary antibodies conjugated to Alexa-488 or Alexa-568 (Abcam; 1:500) and stained with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma, 1:1000). Slides using Iba1, Tmem119, and/or APC antibodies were boiled for 3 minutes in citrate buffer for antigen retrieval prior to the block step.