Paraffin-embedded tissue sections at thickness of 8 μm were used in this study. Coronal sections were prepared from 1 mm behind the bregma. For immunostaining, the following primary antibodies were used: anti-CD4, C-Terminal antibody produced in rabbit (1: 200, SAB4503583, Sigma-Aldrich), Purified Rat Anti- Mouse CD8a (1: 200, 550281, BD Biosciences), Alexa Fluor® 647 anti-mouse CD19 Antibody (1: 100, 550281, BioLegend), Purified Rat Anti-Mouse Ly-6G (1: 200, 550291, BD Biosciences), Alexa Fluor® 488 anti-mouse CD45.2 Antibody (1: 100, 109815, BioLegend). Primary antibodies were incubated at 4°C overnight, followed by incubation with species-specific Alexa Fluor (488 and 594)-conjugated secondary antibodies for 1 h. Pictures were acquired with a Nikon Eclipse T300 fluorescence microscope and analyzed using Image Pro Plus (Media Cybernetics, Inc. Rockville, MD). For cell counting, positive cell numbers were counted in every tenth tissue section through the entire tissue block (25 (link)–27 (link)).
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