Anti type 4 collagen

Vessel morphology was analyzed on 4 consecutives 20 μm thick frozen brain sections sampled on 8 animals (four 9 L gliomas and four C6 gliomas). Briefly, after fixation and saturation, brain sections were incubated overnight at 4 °C with primary antibodies to anti-type-IV collagen (Southern Biotech, Clinisciences, Montrouge, France, 1/1000) then with horseradish peroxidase labeled extravidin. Colour was developed with 0.5% (w/v) 3,3-diaminobenzidine (DAB), 0.03% (v/v) hydrogen peroxide followed by counterstaining with hematoxylin. The sections were scanned using a slice scanner at 10X using a Zeiss axio scan system. Quantitative analysis was performed using an automatic segmentation with 7 processing steps (subtract background, colour deconvolution, make binary, remove outliers, despeckle binary, close binary, and fill holes binary) using the freely available image-processing program FIJI (an ImageJ distribution; https://fiji.sc/)18 (link). Vessel characterization within 2 regions of interests (ROI; tumor and healthy striatum) was performed using Matlab (extraction of minor and major vessels length and vessels area; The MathWorks Inc., Natick, MA, USA). Then metrics from each of the 4 slides per animal were pooled. Finally we computed the BV_histo and the VSI_histo for both ROIs using formulae previously published14 (link).

Animals were euthanized 2 weeks after ischemic onset. Brains were quickly removed and frozen, and coronal sections of 20 μm thickness were prepared. Infarction volumes were quantified on Nissl-stained sections using the “indirect” morphometric method with Image J software. Immunohistochemistry was performed as described before (Esposito et al., 2013 (link)) using primary antibodies anti-Doublecortin (DCX) (1:100, #18723 Abcam) and anti- BrdU (1:200, B35130 Invitrogen) as a marker of neurogenesis, and anti-type IV Collagen (1,10, #1340–01 SouthernBiotech) for vascular remodeling and anti-Ki67 (1,500, #1667 Abcam), as proliferation marker, for detecting angiogenesis. GFAP (1,200, #130300 Invitrogen), as a marker of astrocytes, PDGFβ (1,200, #AF1042 R&D systems), as marker of pericytes, Iba1(1,200, #019–19,741 WAKO or Abcam 5,076), as marker of microglia, or NeuN (1,200, #MAB377 Millipore) as marker of neurons, were co-stained with BDNF (1,200, # ab46176 Abcam) or VEGF (1,50, #sc152 Santa Cruz). Three nonoverlapping areas (0.125 mm² per area) were chosen in the boundary zone of the ischemic core to analyze the peri-infarct area.