Bz h1m analyzing system

Retinal sections (7 μm thick) fixed in 4% paraformaldehyde (32 (link)) or RPE-choroid flat mounts (12 (link)) were prepared and incubated with 0.1% Triton X-100 in PBS and 10% normal goat serum (Dako, Carpinteria, CA), as previously described. Then the samples were incubated with primary antibodies followed by Alexa Fluor secondary antibodies (Invitrogen). The primary antibodies were: anti-Angptl2 (ab35574; Abcam), anti-Rpe65 (MAB5428; Merck Millipore), anti-F4/80 (Cl:A3-1; AbD Serotec, Raleigh, NC), anti-MCP-1 (ab8101; Abcam), and anti-Ly5.1 and anti-Ly5.2 (110718, 109816; Biolegend, San Diego, CA). The sections were examined under a microscope equipped with a digital camera (Olympus Co., Tokyo, Japan) or a BZ-H1M analyzing system (Keyence, Osaka, Japan). The immunofluorescence intensity of F4/80 was calculated by summing the fluorescent areas using ImageJ software (National Institutes of Health, Bethesda, MD).

SCD-1 expression in LG histological sections from both wild-type and SCD-1 KO mice was detected immunohistochemically. The isolated LG were embedded in optimal cutting temperature (OCT) compound (Sakura Finetechnical, Tokyo, Japan) and fresh-frozen on ice. 5-μm thick frozen sections on glass slides were fixed with 10% neutral buffered formalin (NBF) (Wako, Osaka, Japan). Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 10 minutes. The glass slides were blocked with 10% bovine serum albumin (BSA) and incubated with mouse monoclonal primary antibody against SCD-1 (1:1000; Cell Signaling Technology, Beverly MA, USA) overnight at 4 °C. Following washes with Tris-Buffered Saline + Tween-20 (TBST), sections were incubated with rabbit anti-mouse IgG antibody conjugated to Alexa 555 (Dako, Glostrup, Denmark). Nuclear staining was performed with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Dojindo, Kumamoto, Japan). Immunofluorescent signals were detected by viewing samples in a fluorescent microscope (BZ-H1M analyzing system; Keyence, Osaka, Japan).