Hoestch 33342

ZO–1, Thermo Fisher Scientific, catalog number (33–9100), lot number (TL277395), clone number (zo1–1A12);
MyoVIIa, Proteus, catalog number (25–6790), lot number (110119), clone number (not available);
Neurofilament H, Sigma–Aldrich, catalog number (AB5539), lot number (3328929), clone number (not available);
Dexamethasone, Abcam, catalog number (ab35000), lot number (GR246267–19), clone number (not available);
Hoestch 33342, Thermo Fisher Scientific, catalog number (H3570), lot number (1387197), clone number (not available).

Another set of brain sections was blocked in 10% normal goat serum/0.05% Tween-PBS (blocking buffer) 1 hour at room temperature and incubated at 4°C overnight with primary antibodies that identified glioma and proliferating cells, respectively: mouse Anti SOX2 (1 : 50) (sc-365964, Santa Cruz Biotechnology, Dallas, TX) and rabbit Anti-Ki-67 (1 : 400) (Ab9260, Chemicon International, Temecula, CA) in blocking buffer. The antibodies were removed and the sections were washed three times with 0.05% Tween-TBS for 10 minutes and then incubated 1 hour at room temperature with secondary antibodies: Alexa 594 A-21203 (1 : 500) (Life Technologies, Carlsbad, CA) and FITC sc-2078 (1 : 500) (Santa Cruz Biotechnology, Dallas, TX). Nuclei were stained with Hoestch 33342 (Thermo Scientific, Waltham, MA). Sections were covered from light, washed, mounted with Fluoro Care Anti-Fade Mountant (Biocare Medical, Concord, CA), and visualized in an Olympus Bx43 fluorescence microscope. The tumor area and its infiltration length were quantified by using the program Image-Pro Plus 7.0 Media Cybernetics (Rockville, MD). The considered tumor area was the largest one of all the sections obtained from each brain, and the infiltration length was measured from the implant site to the longest distance reached by astrocytoma cells.