Gfap cy3 antibody

Cells were washed in PBS, fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton-X 100, washed, incubated in 10% goat serum for 1 h at RT, then incubated with a GFAP-Cy3 antibody (Sigma-Aldrich, #C9205) or HMGB1 antibody (Abcam, #ab18256) at 4° C overnight. After washing, slides were incubated with Alexa conjugated secondary antibodies for 30–45 min at RT. Nuclear DNA was stained with DAPI in mounting media (Vectasheild).

Immunostaining was performed using the previously established protocol in our lab [19 (link)]. Frozen 25 μm coronal sections encompassing the entire tumor were generated from each mouse brain. The sections were blocked with 5% normal goat serum/5% normal horse serum (Vector lab., Burlingame, CA) in PBS containing 0.3% Triton X-100 and 0.05% Phenylhydrazine for 30 minutes and then incubated with monoclonal mouse anti-Pyk2 antibody, dilution 1:1000 (Cell Signaling; #3480S), polyclonal rabbit anti-Iba1 antibody (Wako; #019–19741) 1 μg/ml, and monoclonal GFAP-Cy3 antibody produced in mouse (Sigma, #C9205), dilution 1:500, in PBS-TAT (0.3% TritonX-100, 5% normal goat/5% normal horse serum, 1% sodium azide, 0.01% thimerosal) overnight at 4°C. The sections were incubated with corresponding secondary antibodies (AMCA anti-mouse IgG and fluorescein anti-rabbit IgG (Vector Lab., Burlingame, CA)) overnight and visualized using an Olympus Fluoview FV1000 confocal microscope with 10× objectives or 40× oil immersion objectives.