Alexa fluor 488 conjugated goat anti rabbit second antibody

The subcutaneous pancreatic tumor-bearing C57BL/6 mice were treated as above described. After treatments for 72 h, the mice were euthanized to extract tumors and the collected tumors were cut into 10-μm sections for immunofluorescence staining using anti-PD-L1 antibody (dilution 1:100, Abcam Inc., cat. no. ab213480) and Alexa Fluor 488-conjugated goat anti-rabbit second antibody (dilution 1:200, Abcam Inc., cat. no. ab150077).

Cell culture medium was replaced with 5% goat serum to block non-specific reaction at room temperature for 30 min and then the cells were incubated with 1:500 diluted rabbit anti-α-SM-actin primary antibody (Abcam, UK) for 6 h at room temperature. Then the cells were washed three times with 0.01 M PBS and incubated with 1:500 diluted Alexa Fluor 488-conjugated goat anti-rabbit second antibody (Abcam, UK) in the dark for 3 h at room temperature. Hoechst33342 was used to stain the cell nuclei in the dark for 0.5 h at room temperature. The α-SM-actin immunopositive cells were observed and photographed with a fluorescent microscope (Leica DMIRB, Germany).