Ab36999

MSCs and OPCs were fixed with 4% paraformaldehyde for 10 minutes and incubated 1h in blocking and permeabilization solution [0.1% Triton X-100 (Sigma) and 5% normal goat serum (MP Biomedicals) in phosphate-buffered saline (PBS)]. Primary antibodies, diluted 1:500 in blocking/permeabilization solution, were incubated overnight at 4 °C and detected using antirabbit and -mouse secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen; 1:250 dilutions; 2h incubations at room temperature). To identify cell nuclei, samples were counterstained with 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI) in PBS. The primary antibodies used for immunofluorescence were against the A2B5 antigen (Millipore, MAB312), galactocerebroside (GalC ; Millipore, Ab142), NuMA (Abcam, ab36999), and topoisomerase II-α (Abcam, ab75765).

According to an approved animal protocol (University of Wisconsin Institutional Animal Care and Use Committee), NOD SCID mice whose brains were implanted with 200,000 GSCs from GSC lines 12.1, 22, 44, and 107 were euthanatized for paraffin embedding, and 5-µm thick tissue sections were obtained in the UW Experimental Pathology Shared Service Translational Research Initiatives in Pathology (TRIP) laboratory. Masson’s trichrome staining was performed by UW Carbone Cancer Center experimental pathology shared services on paraffin-embedded samples. For Picrosirius red staining, sections were deparaffinized in xylene and hydrated through decreasing grades of alcohol. These sections were incubated in 0.1 % (wt/vol) Sirius red F3B (Bayer chemicals) in saturated Picric acid solution for 1 hour at room temperature and then rinsed with distilled water, and stained with Mayer’s haematoxylin, followed by differentiation in 1% Hcl, alkalinization with tap water, dehydration, and final mounting. Sections were then examined using circular polarized light. The same methods of staining were used for TMA slides. Immunohistochemistry was performed as previously described^{28 (link)}. A human specific anti-NUMA antibody (ab36999, Abcam) was used at the concentrations of 1:200 on mouse xenograft slides.

Paraffin-embedded tissues were cut into 5 μm sections and dried before use. To begin, sections were deparaffinized in xylene and rehydrated in graded ethanol solutions. Antigen retrieval was performed by boiling in 10 nM sodium citrate buffer (pH 6.0) for 20 min. Washing steps were performed with 1×PBS and primary antibodies were incubated at 4°C overnight in a humidified chamber. All primary antibodies (BrdU, Abcam, ab6326, 1:1000; NuMA, Abcam, ab36999, 1:250) were diluted in 5% BSA, 0.5% Tween-20 blocking buffer.