Egf receptor e746 a750 specific rabbit mab antibody

After SPECT imaging, the xenografts were sacrificed immediately and the tumor tissues were harvested. The tumor tissues were extracted in modified RIPA buffer on cold ice and the concentration of each sample was measured with a BCA protein assay kit (Pierce Biotechnology, Inc). Total proteins were loaded and separated by SDS-PAGE (10%), transferred to a nitrocellulose membrane and then incubated with 5% non-fat milk blocking buffer at room temperature for 30min. The membrane was then incubated at 4°C overnight with primary antibodies including EGF Receptor(D38B1) Rabbit mAb antibody or EGF Receptor (E746-A750 Specific) Rabbit mAb antibody, which were purchased from Cell Signaling Technology and this was followed by incubation with anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology) at room temperature for 1 hour. The bands were detected by an ECL western blotting detection system (BD).

Immunofluorescence staining was performed to determine the total EGFR and EGFR exon 19 E746-A750 deletion expression levels. Tumor tissues were frozen overnight at −80°C, then cut into 5 μm per slice, fixed with 4% cold paraformaldehyde (PFA) for 30 min and washed three times in PBS, then blocked with 10% normal goat serum for 1 h. The slices were then incubated with EGF Receptor (D38B1) Rabbit mAb antibody (1:50, Cell Signaling Technology) or EGF Receptor (E746-A750 Specific) Rabbit mAb antibody (1:100, Cell Signaling Technology) at 4°C overnight, followed by incubation with Goat anti-Rabbit IgG (H + L) or Alexa Fluor^® 594 conjugate (1:1000, ThermoFisher) secondary antibodies and washed five times with PBS buffer. The slices were mounted with 49-6-diamidino-2-phenylindole (DAPI)-containing mounting medium and observed in a confocal microscope (C2Si, Nikon).