Vectashield antifade mounting medium with dapi h 1200 10

Tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Alexa Fluor 488 conjugated goat anti-mouse IgG cross-adsorbed secondary antibody (2 mg/mL, Cat #A32723) and paraformaldehyde were obtained from Thermo Fisher Scientific Inc. (Rockford, IL, USA). Phosphorylated EGFR monoclonal IgG1 mouse antibody (15A2:sc-81488) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Annexin V binding buffer 1×, APC Annexin V, 7-Amino-Actinomycin D (7-AAD) and PI/RNase staining buffer were obtained from BD Biosciences (San Jose, CA, USA). The secondary goat anti-mouse IRDye secondary antibody was purchased from LI-COR Biotechnologies (Lincoln, NE, USA), and the Vectashield Antifade Mounting medium with DAPI (H-1200-10) was purchased from Vector Laboratories (Burlingame, CA, USA). CIEAs were provided by Dr. Halaweish (South Dakota Ste University, Brookings, SD, USA). All other chemicals used were of analytical grade.

Paraffin sections were deparaffinized, washed in PBS, and subjected to antigen retrieval. Sections were incubated with the primary antibody at 4°C overnight, then with the secondary antibody for 2 h at room temperature (RT), then mounted with VECTASHIELD Antifade Mounting Medium with DAPI (H-1200-10; Vector Laboratories, Burlingame, CA, USA). We used the following primary antibodies: anti-phospho-Akt (Ser473)(#9271, Cell Signaling Technology) and anti-phospho-Erk1/2 (Thr202/Tyr204)(#9101, Cell Signaling Technology). Representative images were captured using a fluorescence microscope (Leica).