Enucleated eyes were fixed in formaldehyde (Super Fix, Kurabo, Osaka, Japan) for 7 days and then embedded in paraffin (Sigma-Aldrich) [20 (link),21 (link)]. paraffin sections were sliced into 10 µm-thick sections and prepared as a series of five sequential slides with an autoslide preparation device (Kurabo). We first performed H&E staining before IHC evaluation. Retinal sections were blocked with 5% goat serum in PBS for 1 h at room temperature. Primary antibodies to the following antigens were applied: CD3 (host: rabbit, Abcam, Cambridge, UK), Iba1 (host: rabbit, Wako, Osaka, Japan), MHC class II (MHC-II; host: mouse, DakoCytomation, Glostrup, Denmark), CD20 (host: rabbit, Abcam), immunoglobulin G (IgG; host: rabbit, Abcam), NKG2A (host: rabbit, Abcam), and IFN-γ (host: mouse, R&D Systems, Minneapolis, MN, USA). All of the antibodies were incubated at 4 °C overnight. After washing, sections were incubated with secondary antibodies for 1 h at RT and counterstained with DAPI (Life Technologies, Carlsbad, CA, USA). Images were acquired using a confocal microscope (LSM700, Zeiss, Jena, Germany) [20 (link),21 (link)].
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