Anti mouse igg af647

Single-cell suspensions were prepared and splenocytes were incubated with APC-conjugated anti-CD4 (0.25μg/10⁶ cells in 100μl volume, 100412 BioLegend) and Perp-Cy5.5-conjugated anti-CD8 (1μg/10⁶ cells in 100μl volume, 100733 BioLegend) for 30 min on ice. For HLA class I analysis, cells were counted and 2×10⁶ cells were incubated with mouse anti-HLA class I antibody (1:200, 311402 BioLegend) for 30 min followed by secondary antibody (anti-mouse IgG AF647, Thermo Fisher) incubation for 30min. Cells were analyzed by flow cytometry on LSRII or LSRFortessa device with BDFacs DIVA software. The data were analyzed by Flow Jo software (TreeStar).

To collect the brain samples, engrafted mice were euthanized on the indicated analysis date. Tissue was fixed in 4% PFA for 1 day and then held in 30% sucrose for 2 days at 4°C. Thick sections (150 μm) were prepared by a Vibratome (VT1200; Leica, Wetzlar, Germany) and then incubated in blocking solution (15% normal goat serum/PBS) for 1 h at RT. Sections were incubated in anti-mCD31 (3528: Cell Signaling Technology, Danvers, MA, USA) or anti-mGFAP (ab4674; Abcam) and anti-hVEGF-AA (sc-152; Santa Cruz Biotechnology, Dallas, TX, USA) or anti-hIL-15 (ab55276; Abcam) in blocking solution overnight at 4°C, washed 3× in PBS, and incubated in anti-mouse IgG-AF647 (A21237; Thermo Fisher), anti-rabbit-IgG-AF555 (A32732; Thermo Fisher) or anti-chicken IgG-Rhodamine (703-295-155; Jackson ImmunoResearch) at 1:2000 in blocking solution for 1 h at RT. After 3 washes, sections were mounted with Prolong Gold Antifade Mountant with DAPI (P36935; Thermo Fisher) for imaging by a confocal microscope. For IHC of formalin-fixed paraffin-embedded brain, 5-μm slices were mounted on charged slides, deparaffinized, and stained on the Ventana Discovery Platform (Roche) with anti-hCD10 antibody (ab82073; Abcam) and anti-rabbit HQ (760–4815; Roche).