Anti nestin ab22035

Seventeen days post-inoculation, C6 GSC-derived subcutaneous tumors were removed, fixed in 3.7% paraformaldehyde (Sigma-Aldrich, Darmstadt, Germany), dewaxed with xylol, and rehydrated using alcohols in decreasing concentration. Paraffinated tumors were sectioned (5 μm) and mounted on silanized slides. Then, samples were dewaxed with xylol, rehydrated using alcohols in decreasing concentration, and immersed in heated citrate buffer (pH 6) for antigen retrieval. Endogenous peroxidase was blocked using 3% hydrogen peroxide, followed by incubation with 5% BSA for 1 h and incubation with anti-MRP1 (sc-18836; Santa Cruz Biotechnology), anti-Ki67 (sc-15402; Santa Cruz Biotechnology), anti-nestin (ab22035, abcam, Cambridge, MA, United States), and anti-GFAP (sc-33673, Santa Cruz Biotechnology) antibodies. Finally, immunodetections were detected using the ImmPRESS HRP Universal Antibody Polymer Detection Kit (Vector Laboratories, Burlingame, CA, United States) with Liquid DAB + Substrate Chromogen System, K3468, DAKO (Santa Clara, CA, United States) following the manufacturer′s instructions and using hematoxilin as a counterstain. Samples were visualized using a light microscope (Zeiss) and 10 images (20×) were processed using ImageJ software (NIH), considering the values of vehicle treatment as a calibrator, and normalized to 100%.