Anti glial fibrillary acidic protein

Hanks fluid (Leagene,CC0033), glycerin (Sigma,G9012), SP Rabbit HRP Kit (DAB) was brought from Cwbiotech (CW2035S), HRP-labeled Goat Anti-Mouse IgG (H+L) was purchased from Beyotime Biotechnology (Shanghai, China), Antibodies were obtained from the following sources: anti-HLA-A (Abcam, ab52922), anti-CD45 (Abcam, ab10558), anti-Ki67 (Abcam, ab15580), anti-glial fibrillary acidic protein (Dako), anti-Human Carcinoembryonic Antigen (clone II-7, Dako).

To stain 2D surfaces glioblastoma TICs, cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes and permeabilized with 0.25% Triton X-100 for 10 min and blocked with 5% goat serum for 1 hour. The samples were then incubated with primary antibodies at room temperature for 2 hours. After washed with PBS for 3 times, secondary antibodies in 2% BSA were added and incubated for another 1 hour before imaging. To stain 3D fibrous glioblastoma TICs in AlgTubes, cells were fixed with 4% PFA at room temperature for 1 hour, then incubated with PBS+ 0.25% Triton X-100+ 5% goat serum+ primary antibodies (Nestin, 1:200, Millipore; SOX2, 10 µg/mL, R&D system; Olig2, 20 µg/mL, Novus Biologicals; Tuj1, 1:10,000, Sigma; anti-glial fibrillary acidic protein, 1:500, Dako; Ki-67, 1:500, Invitrogen) at 4 °C for 2–3 days. After extensive washing, secondary antibodies (Alexa 488 Donkey anti-mouse, 1:500; Alexa 594 Donkey anti-rabbit, 1:500) in 2% BSA was added and incubated at 4 °C for 1 day. Cells were washed with PBS before imaging with NIKON A1 Confocal Microscopy. LIVE/DEAD^® cell viability staining was utilized to assess live and dead cells according to the product manual.

Protein extraction from cells or tissue was performed using a lysis buffer that contained a protease inhibitor cocktail (Complete, Roche Diagnostics, cat. no. 04574834001). Total protein (25 μg) was loaded for each sample for separation by SDS–polyacrylamide gel electrophoresis (8%) and transferred onto nitrocellulose membranes (BioRad; cat. no. 1620115). Membranes were blocked with 5% non-fat milk diluted in Tris-buffered saline containing 0.1% Tween-20 (TBST) for one h and incubated in the presence of anti-Th (Abcam ab112 monoclonal anti-rabbit 1:1000), or anti-Th F-11 (sc-25269 monoclonal anti-mouse, 1:1000), or anti-Th Millipore ab152 (monoclonal anti-rabbit 1:1000), anti-Glial Fibrillary Acidic Protein (DAKO Z0334 polyclonal anti-rabbit 1:2000), anti-Beta-actin-peroxidase (Sigma A3854 monoclonal anti-mouse 1:25000). Proteins were revealed using secondary peroxidase-coupled anti-rabbit and anti-mouse antibodies (Jackson ImmunoResearch), using the Western Lightning Plus-ECL Kit (PerkinElmer; Waltham, MA). Images were digitally captured using the FUSION SOLO S instrument (Vilbert Smart imagining). Autoradiograms were analyzed by densitometry using the Image J^® software (NIH).