Rabbit mab antibody

After radioactivity decay following the PET/CT studies, the mice were sacrificed and the tumor tissues harvested for western blots. Tumor tissue proteins were extracted with extraction buffer and their concentration was determined using a BCA protein assay kit (Pierce Biotechnology, Inc.). After sodium dodecyl sulfate polyacrylamide gel electrophoresis separation of 90 ug of total protein, the proteins were transferred to a nitrocellulose filter membrane (NC) membrane and incubated at room temperature with 5% nonfat milk blocking buffer. The blots were then incubated overnight at 4 °C with EGF Receptor (D38B1) Rabbit mAb antibody (1:1000 Cell Signaling) and EGF Receptor (E746-A750 Specific) Rabbit mAb antibody (1:1000; Cell Signaling), followed by incubation at room temperature for 2 h with anti-rabbit IgG, HRP-linked antibody (Cell Signaling) or anti-mouse IgG, HRP-linked antibody (Cell Signaling) in 5% BSA/TBST. The protein bands were detected using the ECL Western blotting detection system (BD). GAPDH was used as a loading control. After development, the films were scanned with BIO-RAD Gel Doc XR+. The images were opened and analyzed by ImageLab(BIO-RAD) software. Three samples of each tumor model were prepared for western blot to obtain semiquantitative data for statistical analysis.

Frozen tumor tissue slices (5 μm) were fixed with cold 4% paraformaldehyde (PFA) for 30 min, and then washed with PBS three times. After blocking with TBS 0.1% Tween-20 (TBST)/10% normal goat serum for 1 h, the sections were incubated with EGF Receptor (D38B1) Rabbit mAb antibody (1:50; Cell Signaling) and EGF Receptor (E746-A750 Specific) Rabbit mAb antibody (1:50; Cell Signaling) at 4 °C overnight and then covered with fluorescein isothiocyanate-conjugated (FITC) secondary antibody Goat anti-Rabbit IgG (H + L) Secondary Antibody, Alexa Fluor® 594 conjugate (1:1000; ThermoFisher). After being washed three times with PBS, the slices were mounted with 49-6-diamidino-2-phenylindole (DAPI)-containing mounting medium and observed under a confocal microscope. Each type of tumor tissue slices was prepared in triplicates.