Fluorophore labeled secondary antibodies

Tissue hypoxia was detected using the Hypoxyprobe-1 Omni Kit (Hypoxyprobe Inc. Burlington, MA, United States) as described previously (Liu et al., 2015 (link); Sun et al., 2016 (link)). Briefly, mice were injected (i.p) with pimonidazole (60mg/kg). After 30min, tissues were collected, fixed overnight in 4% PFA, and embedded in paraffin. Paraffin sections (5μm) of the heart, lungs and kidneys were deparaffinized, rehydrated, and endogenous peroxidase was blocked with 0.3% H₂O₂. After heat-induced antigen retrieval, the sections were incubated with anti-hypoxyprobe antibody (1:200) overnight at 4°C. The bound antibodies were detected with 1) fluorophore-labeled secondary antibodies (1:1,000, Jackson ImmunoResearch Lab). Nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI) (sigma-Aldrich); or 2) the biotin–streptavidin–peroxidase system (UltraSensitive-SP-kit, MaiXin Biotechnology, Fuzhou, China) using diaminobenzidine (Sigma-Aldrich) as the chromogen. Counterstaining was performed with hematoxylin.

Mice were humanely euthanized, and eyes enucleated and immersion-fixed in 4% paraformaldehyde (PFA) for 10 min. Eyeballs were then rinsed in PBS, and eye cups were generated by removing the anterior segment. The eye cups were infiltrated in 30% sucrose overnight and embedded in Tissue-Tek OCT (Sakura Finetek, Torrance, CA, USA). Immunostaining for CD59a was performed on 10-μm-thick cryostat sections. The primary antibodies were mouse anti-mouse CD59a (HM1116, Hycult, PA, USA) at 1:500 dilution and a mouse-on-mouse polymer IHC kit (ab127055, Abcam, MA, USA) was used to optimize signal to background staining ratio. We used an HRP-conjugated secondary antibody followed by diaminobenzidine to yield a brown precipitate. Rat anti-mouse glial fibrillary acid protein (GFAP) antibody (13–0300, Invitrogen, Camarillo, CA, USA) was used at a 1:500 dilution. GFAP antibody was detected using fluorophore-labeled secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., PA, USA). Control sections were treated identically but without primary antibody. The sections were analyzed by fluorescence microscopy with identical exposure parameters (model TE300 microscope, Nikon, Tokyo, Japan) with ImagePro software (Media Cybernetics, Silver Spring, MD, USA).