Rat 401

Frozen brain sections (8-10 μm) were fixed in 2% paraformaldehyde, blocked with Power Block (Biogenex), and incubated with rabbit anti- glial fibrillary acidic protein (GFAP; 1:100; Cell Signaling Technology, Danvers, MA), mouse anti-nestin (1:10; Rat-401; Developmental Studies Hybridoma Bank, University of Iowa) or rabbit anti-Ki-67 (ScyTek) for 24 hs at 4ºC. Alternatively, frozen sections were air-dried, blocked with 5% normal goat serum, and incubated with mouse anti-phosphatidylserine antibody (1:100; clone 4B6; Abcam); rat anti-CD11b (1:10; M1/70.15.11.5.2; Developmental Studies Hybridoma Bank); rabbit anti-vascular endothelial growth factor receptor 1 (VEGFR1; 1:100; Bioss, Woburn, MA), or mouse anti-bovine lactadherin (1:5; L688; Developmental Studies Hybridoma Bank) for 24 hs at 4ºC. Species-specific, Alexa-488 or Alexa-555-conjugated, goat secondary antibodies (Cell Signaling Technology) were used to label primary antibodies. A biotinylated anti-rabbit antibody followed by Streptavidin-FITC staining was used to detect VEGFR1. Incubations were carried on in PBS containing 25% glycerol. Microphotographs were taken with either a LSM 710 confocal microscope or an Olympus BX51epifluorescence microscope.

NSCs exposed to HS and subsequently allowed to recover, along with cells exposed to 37°C, were evaluated using immunofluorescence staining. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature, treated with 0.5% Triton X-100 in PBS, blocked with 10% bovine serum albumin (BSA) in PBS for 60 min at room temperature, and washed 3 times for 5 minutes with PBS. The cells were incubated with the primary antibody for 60 min and washed with PBS. The primary antibody used was anti-nestin (Rat-401, 1:100, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), a marker of NSCs. The cells were subsequently incubated with Alexa Fluor546-labeled secondary antibodies (1:200, Molecular Probes, Eugene, OR, USA) for 30 min. The nuclei were counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride. Cells were viewed under a fluorescence microscope (BH-2; Nikon) and photos were analyzed with Photoshop Elements 12 (Adobe, CA, USA).

For organotypic co-culture, young adult (5 weeks old) C57BL/6 SCRM (BL6) mice were used. They were sacrificed by cervical dislocation and processed as previously described in (Marques-Torrejon et al., 2018 (link)). Samples were blocked in 10% normal goat serum and 0.2% Triton X-100 in PBS for 1 h and incubated for 48 h in blocking buffer with the appropriate primary antibodies: GFAP (1:500, Z0334 DAKO) KI67 (1:100 Thermo RM9106), SOX2 (1:100, AB5603, Millipore), NESTIN (1:10, Rat 401, Developmental Studies Hybridoma Bank), p53 (1:500, ab6326, Cell signalling), LRIG1 (1:100, R&D, AF3688), CD9 (1:100, 14–0091–82, eBioscience, 1:500), RFP (1:500, Abcam 62,341), BrdU (1; 1,000 Abcam, ab6326), SSEA1 (1:500, Biolegend, ab63260), Vimentin-40E (1:50, DSHB), CD133 (1:500, Milllipore, ab6326), CD9 (1; 500,14–0091–82, eBioscience), and pSMAD1/5 (1:500, Cell signalling, 9,516).