C2 upright confocal microscope

Mice were euthanized, and eyes collected and fixed in 2% paraformaldehyde for 1 hr before transferring into cold PBS. For staining of endothelial or pericyte markers eyes were fixed in ice cold methanol. After dissection of the various compartments of the eye (choroid, retina, iris) the tissues were incubated in 20 mM EDTA at 37°C for 30 minutes. Tissues were then incubated in a solution containing 0.3% Triton-X, 2% bovine serum albumin and 10% of normal goat serum in PBS at room temperature for 30 min. Tissues were incubated with the primary antibody overnight at 4°C followed by incubation with the secondary antibody at room temperature for 1 hr. Detection of the immediate-early (IE1) protein of MCMV was performed with the 6/58/1 monoclonal antibody [53 (link)]. Monoclonal antibodies specific for the following cell surface markers were used to stain tissue sections: MHC-class II (clone M5/114), CD45 (clone 30F11), CD31 (clone Mec 13.3), PDGFRβ (clone APB5), F4/80 (clone BM8), CD4 (clone RM4-5), CD8 (clone 53–6.7), IBA1 (Wako Pure Chemicals Industry, Osaka, Japan), CollagenIV (Biorad-2150-1470). Slides were counterstained with Hoechst to visualize nuclei. Assessment of stained specimens was performed using an epifluorescence microscope (Olympus BX60 microscope: Olympus, Tokyo, Japan) or a Nikon C2 Upright Confocal microscope.