Mouse anti human frat1 monoclonal antibody

Intracranial gliomas of all mouse groups were dissected and post-fixed in 10% formalin, followed by conventional paraffin-embedding, tissue sectioning into 4 µm thickness, and mounting to polylysine coated slides. After high-pressure heat retrieval of antigens, each tissue section was quenched in 3% H₂O₂ and washed in PBS for three times, followed by blocking antigen with non-immune serum for 30 min and incubating tissue sections with 1:50 mouse anti-human FRAT1 monoclonal antibody and 1:1000 mouse anti-human β-catenin monoclonal antibody (Abcam) at 4°C overnight. After three PBS washes, tissue sections were incubated with secondary antibody at 37°C for 30 min and developed in DAB solution (Sigma-Aldrich), followed by rinsing in distilled water, hematoxylin nuclear-staining, and neutral gum mounting.

Immunofluorescent antibody staining was used to identify CD133, nestin, and FRAT1 expression in GSCs. Cultured GSCs were placed on glass slides coated with 40 g/l polylysine solution and post-fixed in 4% paraformaldehyde for 15 to 20 min, followed by washing three times in 0.01 mol/l PBS for 5 min each. After blocking non-specific antigen on the cell surface using 1% BSA at 37°C for 1 h, cells were incubated with 1:300 PBS-diluted primary antibody at 4°C for 8 h and 37°C for 1 h. Then, cells were incubated with 1:200 PBS-diluted fluorescent secondary antibody in the dark at 37°C for 40 min. Cell nuclei were eventually stained with DAPI solution (Abcam). After washing the stained cells with PBS, cytomorphology was observed with a fluorescent microscope and photographed with a digital camera. Primary antibodies used included rabbit anti-human CD133 monoclonal antibody (Abcam), rabbit anti-human nestin monoclonal antibody (Abcam), and mouse anti-human FRAT1 monoclonal antibody (Abcam). Secondary antibody used included Alexa Fluor 488 (goat anti-rabbit fluorescent antibody, Cell signaling technology, Danvers, MA) and Alexa Fluor 555 (goat anti-mouse fluorescent antibody, Cell signaling technology).