Anti mouse igg alexa flour 488

Astrocytes plated on glass coverslips were treated with andrographolide, then fixed with 4 % paraformaldehyde/PBS for 15 min, and washed thrice with PBS followed by incubation with permeabilizing buffer containing 0.1 % Triton-X100 in PBS for 5 min at 25 °C. The cells were then incubated in blocking solution (5 % BSA in permeabilizing buffer) for 1 h before incubation overnight with primary antibodies against HO-1 (1:200 dilution) in blocking solution at 4 °C. Subsequently, cells were washed thrice with PBS and incubated with anti-mouse IgG Alexa Flour® 488 (1:400 dilution, Cell Signaling Technology, Danvers, MA, USA) for 1 h at 25 °C. Cells were then washed with PBS before mounting the coverslips onto glass slides using mounting medium containing DAPI nuclear stain (Vector Laboratories, Burlingame, CA, USA). Immunofluorescence images were taken with an Axioplot microscope equipped with Carl Zeiss 510 confocal imaging scan-head and software (Carl Zeiss MicroImaging, Jena, Germany), with the same parameters used for all images.

Lysates were reduced, boiled, subjected to 4-20% SDS-PAGE and transferred to LF-PVDF membranes. Membranes were blocked with 3% BSA and incubated with pSTAT3 (Abcam ab76315), STAT3 (Cell Signaling 4904) or B-Actin (Cell signaling 3700) at 4°C overnight. Membranes were then probed with either anti-mouse IgG Alexa Flour 488 (Cell Signaling 4408) or anti-rabbit IgG Alexa Fluor 647 (Cell Signaling 4414) for 1 h at room temperature. Membranes were visualized using the Chemi-Doc MP System (BIO-RAD).