Anti human nuclei

Immunohistochemistry was performed as previously described [44] (link). Briefly, injected animals were perfused at different time points with 4% paraformaldehyde (PFA). The brains were removed and immersed in 4% PFA overnight at 4°C and then equilibrated in 30% sucrose. Entire brains were processed in sagittal 40 microns microtome sections. Sections which were positive with GFP-labelled cells were blocked with 5% donkey serum, permeabilized with 0.25% Triton X in TBS for 30 minutes before application of primary antibodies of rabbit anti-GFAP (Dako, Glostrup, Denmark), anti-PDGFRα (Millipore), goat anti-doublecortin (Santa Cruz, CA, USA), mouse anti-human nestin (Millipore) and anti-human nuclei (Millipore) at dilutions of 1∶100 to 1∶500 for overnight incubation at 4°C. Incubations with secondary antibody (1∶500) of 647 donkey anti-rabbit or 647 donkey anti-goat with 555 donkey anti-mouse for one hour at room temperature were performed, followed by a five minute staining with DAPI (Millipore), before sections were mounted on slides with mounting medium. The staining was viewed using Zeiss LSM 710 confocal system (Carl Zeiss Pte Ltd, Singapore) at 63X magnification.

Ten-micrometer sections from cryopreserved tissue blocks were fixed with 3% PFA for 15 min and incubated overnight at 4 °C with the following primary antibodies: anti-Por (Abcam, cat# ab13513) diluted 1:500, anti-human Nuclei (EMD Millipore, cat# MAB1281) diluted 1:250 in PBS containing 0.2% Triton X-100, and 0.5% BSA. Secondary antibodies (1:1,000 Alexa-fluor conjugated, Molecular Probes) were incubated for 60 min at room temperature in the same buffer. Sections were mounted with Vectashield plus DAPI (Vector Labs).

Immunofluorescence was performed as previously described [9 (link)]. The primary antibodies were: anti-Aurora A/AIK (rabbit monoclonal, 1:100, Cell Signaling Technology®), anti-Phospho-Aurora A (Thr288) (rabbit monoclonal, 1:100, Cell Signaling Technology®), anti-CXCR4 (mouse monoclonal, 1:100, Santa Cruz®, Heidelberg, Germany), anti-Ki-67 (mouse monoclonal, 1:400, BD Biosciences®, Erembodegem Belgium), anti-Human Nuclei (mouse monoclonal IgG, 1:250, Merck Millipore®). The specificity of the used primary antibodies was verified using IgG isotype controls for anti-Aurora A/AIK and anti-Phospho-Aurora A (Thr288) antibodies and secondary antibody incubation alone for other antibodies. The quantification of P-AurA-positive cells was normalized according to AurA/Hoechst double-positive cells per triplicated well for each condition using the Image J software (n = 3).