B35130

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.

Cells grown in YES were synchronized in G1 phase and released in the presence of 10 µM EdU. After the first S phase, EdU was removed by washing the cells three times with equal volumes of YES. Before the second S phase 50 µM BrdU was added and kept in the medium until the second S phase was completed. After adding the analogue the cells were incubated in the dark until they were fixed. Cell fixation, zymolase- and HCl-treatment and blocking were as described above. EdU detection was then performed as described above. Primary antibody against BrdU (Invitrogen cat # B-35130, MoBU1) was used at a dilution of 1∶20 and the cells were incubated overnight at 4°C on a rotating wheel. The next day, the cells were washed 3 times with PBS, 2% FCS and 0.05% Tween-20. Secondary anti-mouse IgG1:FITC (AbD Serotec cat.# STAR132F) was added at a dilution of 1∶250. After incubation for 2 hours at room temperature, the cells were washed 3 times with PBS, 2% FCS and 0.05% Tween-20. The cells were mounted and viewed as above.