Rat anti mouse cd8a

Paraffin-embedded brain tissues were sectioned at 5-μm thickness. Deparaffinization was achieved by washing slides through a series of xylenes and decreasing concentrations of ethanol. Tissue sections were then subjected to antigen retrieval using citrate buffer, pH 6.0 (Sigma-Aldrich) at 95 °C for 30 min. Following rinsing with PBS, sections were incubated in blocking buffer containing PBS with 10% normal donkey serum and 0.03% Triton-X (Sigma-Aldrich) for 2 h at room temperature. Slides were then incubated with primary antibody in blocking buffer overnight at 4°C. The following day, slides were rinsed with PBS then incubated in appropriate Alexa Fluor secondary antibodies (Thermo Fisher Scientific). Sections were then rinsed and stained with Hoechst DNA dye (Thermo Fisher Scientific) before being coverslipped with ProLong mounting medium (Invitrogen). Primary antibodies included rat anti-human CD3 (Abcam), rabbit anti-human CD8α (Cell Signaling), mouse anti-Aβ (Cell Signaling), chicken anti-human MAP2 (Abcam), mouse anti-human granzyme-A (Abcam), rat anti-mouse CD8a (eBioscience) and rabbit anti-mouse NEFH (Abcam). For mouse Aβ plaque staining, ThioflavinS (1 mg ml⁻¹, 1:625, Sigma) was added to the secondary antibody solution.

All antibodies were diluted in 0.2% BSA in DPBS (FACS Buffer). Fc receptors were blocked with purified anti-CD16/32 (eBioscience 14-0161, San Diego CA) at a dilution 1:50 for 15 min at 4 °C. Cells were washed with FACS buffer, and resuspended in a mixture of fluorescently-labeled antibodies at the following dilutions: 1:100 rat anti-mouse CD3 (eBioscience 11-0031), 0.5:100 rat anti-mouse CD4 (eBioscience 47-0041), 0.5:100 rat anti-mouse CD8a (eBioscience 17-0081), 0.5:100 rat anti-human/mouse CD11b (Tonbo Biosciences 11-0112, San Diego CA), 0.5:100 rat anti-mouse CD19 (eBioscience 25-0193), 0.5:100 rat anti-mouse CD44 (eBioscience 25-0441), 0.25:100 rat anti-mouse CD45 (eBioscience 11-0451), 1:100 rat anti-mouse CD127 (eBioscience 12-1271), 1:100 rat anti-mouse F4/80 (eBioscience 50-4801), 0.3:100 rat anti-mouse Ly6c (eBioscience 12-5932), 0.25:100 rat anti-mouse Ly6g (eBioscience 45-5931), and 1:100 rat anti-mouse Siglec F (BD Biosciences 562680, San Jose CA), or appropriate isotype controls for 1 h at 4 °C in the dark. Cells were washed and re-suspended in FACS buffer and run live on the BD Facs Canto II. FLOJO software was used for analysis. Representative dot plots for the gating strategy used to define the various cell types discussed in this manuscript are provided in Fig. 1.