Anti rabbit igg af555

Antibodies used were CD31, AB5690; K5, AB24647; K18, AB668 (Abcam, Cambridge, MA, USA); β-actin, NB600-503SS (Novus Biologicals, Littleton, CO, USA); Angpt1, SC-6320; CD61, SC-19671PE; PCNA, SC-7907; PPARγ, SC-7273 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); CD24-PE, 10814; CD49f-AF488, 60037 (Stem Cell Technologies, Vancouver, BC, Canada); and anti-rabbit IgG-AF488, A11008; anti-rabbit IgG-AF555, A21428 (Thermo Fisher Scientific, Waltham, MA, USA).

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).