Ventana discovery platform

Brains were dissected from the skull and halved. The right hemisphere was embedded in optimal cutting temperature (OCT) media, frozen and sectioned at 5–10 μm. The sections were mounted on positively charged slides, fixed in a 50/50 mixture of formalin/ethanol and then transferred to reaction buffer on the autostainer, the Ventana Discovery platform (Roche, Basel, Switzerland). GFAP (glial fibrillary acidic protein) rabbit polyclonal antibody (Abcam, Cambridge, MA) was diluted at 1:5000 and incubated for 20 min at 36°C. In a second set of slides, Iba-1 (ionized calcium binding adaptor molecule 1) rabbit polyclonal antibody (Abcam, Cambridge, MA) was diluted 1:100 in Discovery PSS and incubated for 32 min at 36°C. The enzyme conjugate (anti-HQ HRP, horse radish peroxidase) was applied for 12 min followed by DAB (3,3’-diaminobenzidine) detection and hematoxylin counterstain with bluing solution. Slides were then removed from the autostainer and coverslipped.

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).