Fluorescence conjugated cross adsorbed secondary antibody

OIR-mice at P17 were euthanized and perfused successively with PBS and 4% PFA, and the intact retinas were collected. Retinas were blocked and permeabilized in PBS containing 10% goat serum and 1% Triton-X-100 (Sigma-Aldrich) for 30 min. Endogenous Fc receptors and IgG were blocked with rat anti-mouse CD16/CD32 (Mouse BD Block^TM, 1:50, BD Biosciences, 553142) and the blocking reagent provided in the mouse-on-mouse kit (Vector Laboratories, Cat. No. FMK-2201, Burlingame, CA, USA), respectively. Retinas were then incubated with primary antibodies against mouse Adora2a (1:100, Millipore, 05-717), rabbit IBa1 (1:400, Sakura Finetek, 019-19741, Torrance, CA), and Alexa488- or Alexa-594 labeled Griffonia simplicifolia isolectin B4 (1:200, Invitrogen, 121411 and 121413, Carlsbad, CA, USA) overnight at 4 °C, followed by incubation with fluorescence-conjugated cross-adsorbed secondary antibody (1:500, Molecular Probes, Life Technologies, A-21131, Carlsbad, CA,USA) for 1 hour, and then counterstained with DAPI (Invitrogen). Retinas were flat mounted on microscope slides in mounting medium (Vectashield; Vector Laboratories) and examined by confocal microscopy (Zeiss 780; Carl Zeiss, Jena, Germany). Areas of vaso-obliteration and vitreoretinal neovascular tufts were quantified using Adobe Photoshop CS 5 software.

Eyes were enucleated and fixed in 4% paraformaldehyde for 2 hours at room temperature. The intact retinas were collected, blocked, and permeabilized in PBS containing 10% goat serum, 3% BSA, 1% Triton X-100, and 0.2% Tween 20 for 1 hour. Samples were then incubated with primary antibodies against rabbit PKM2 (1:100, 4053, Cell Signaling Technology), rabbit Ki67 (1:200, RM-9106, Thermo Scientific), rabbit Fabp5 (1:200, 2.5 μg/mL, RD181060100, BioVendo), rabbit Igf1 (10 μg/mL, AF791, R&D Systems), rat F4/80 (1:100, ab6640, Abcam), rabbit IBa1 (1:400, 019-19741, Sakura Finetek), goat IBa1 (1:400, ab48004, Abcam), rat CD11b (5 μg/mL, 561114, BD Biosciences), and Alexa Fluor 488–, Alexa Fluor 594–, or Alexa Fluor 647–labeled Griffonia simplicifolia isolectin B4 (10 μg/mL; catalogs 121411, 121413, and I32450, respectively; Invitrogen) overnight at 4°C, followed by incubation with fluorescence-conjugated cross-adsorbed secondary antibody (1:500, Molecular Probes, Invitrogen) for 1 hour, and then counterstained with DAPI (Invitrogen). Retinas were flat mounted on microscope slides in a mounting medium (Vectashield; Vector Laboratories) and examined by confocal microscopy (Zeiss 780; Carl Zeiss). For all immunofluorescence experiments, parallel groups of tissues were stained with IgG isotype and secondary antibody as negative controls.