Donkey anti rabbit igg alexa fluor 647

Eyes were enucleated and fixed overnight in 4% PFA at 4 °C. Neural retina (NR) and RPE/choroidal flat mounts were prepared by removal of the anterior segment and separation of the two ocular tissues. Radial cuts from the periphery toward the center were made to flatten the layers. RPE/choroid and NR tissue were incubated in blocking buffer (1% BSA, 0.1% TritonX, 3% donkey serum in PBS) for 1–2 h. Then, the tissues were incubated for 3 days at 4 °C with the following primary antibodies in blocking buffer: Anti-Phalloidin-594 (Invitrogen), Anti-Iba1 antibody (EPR16588, Abcam), Anti-Rhodopsin (Rho 4D2, Abcam), Anti-P2RY12 (AnaSpec, AS-50043A). To distinguish microglia from infiltrating macrophages, retinae from old mice were stained with P2RY12, a microglia-specific marker. After washing with PBS, the tissues were incubated with secondary antibodies (Donkey Anti-Rabbit IgG Alexa Fluor 647 (GR3408408-1, Abcam), Donkey pAb to rabbit IgG (DyLight 650 (GR3349963-2, Abcam), Alexa Fluor 647 Goat anti-Rabbit IgG (A21245, Invitrogen), Alexa Fluor 488 Goat Anti-Mouse IgG (A11001, Invitrogen)) and cell nuclei were stained with DAPI (D9542, sigma) (10 µg/ml for RPE/choroid and 1 µg/ml for NR tissue). The slides were mounted with mounting medium (Polyvinyl alcohol (PVA) with DABCO).

Cells were fixed with 4% PFA in PBS for 15 min, then blocked and permeabilized with 0.3% Triton-X in bovine serum albumin (BSA, 3%) for 15 min. For LAMP-2 immunolabelling, cells were permeabilized with 0.01% saponin as per manufacturer’s instructions, followed by blocking with 3% BSA for 30 min. Cells were incubated with the following primary antibodies overnight at 4 °C; anti-p75ntr (4 µg/ml; BioLegend); anti-S100β (4 µg/ml; ThermoFisher) and anti-LAMP-2 (4.2 µg/ml; Abcam), anti-MBP (4.3 µg/ml; Gene Tex) The following day, cells were incubated with secondary antibodies donkey anti-rabbit IgG Alexa Fluor 488 (4 µg/ml; Abcam), donkey anti-rat IgG Alexa Fluor 647 (4 µg/ml; Abcam) or donkey anti-rabbit IgG Alexa Fluor 647 (4 µg/ml; Abcam; ThermoFisher). Nuclei were stained using Hoechst 33,342 (1 μg/ml, ThermoFisher). High resolution images were obtained using a confocal microscope (Olympus FV3000).

Fibroblast and myofibroblasts were quantified by anti-vimentin and anti-α-Smooth Muscle Actin immunofluorescence-stained OCT tissue sections. 4 µm frozen sections were fixed with 10% formalin, blocked in 10% BSA, 0.1% Triton X-100 TBST, and stained overnight at 4 °C with primary antibodies: rabbit polyclonal anti-αSMA Ab (Abcam, Cambridge, MA) at 1:100 dilution and chicken polyclonal anti-Vimentin Ab (Abcam, Cambridge, MA) at 1:2000 dilution. Secondary antibody at 1:500 dilution was added for 2 h at room temperature with donkey anti-rabbit IgG-Alexa-fluor-647 and goat anti-chicken IgY-DyLight 488 (Abcam, Cambridge, MA). Fluorescent images were captured by the CSMC Imaging Core and analyzed using TissueGnostics TissueFAXS 200 system (Tissue Gnostics GmbH, Vienna, Austria) for cell-based counting of automatically recognized positive cells in a FACS-like manner of scattergram analysis, as described previously^{52 (link),53 (link)}.