Mouse anti myc 9e10 monoclonal antibody

HEK293 stable cells seeded into poly-d-lysine-coated 8-well slides (Biocoat Cellware from Falcon, B&D Systems, Franklin Lakes, NJ, USA) were treated with 0.1% DMSO or 10⁻⁶ M Ipsen 5i for 24 h. On the day of experiment, cells were washed with phosphate buffered saline for immunohistochemistry (PBS-IH, 137 mM NaCl, 2.7 mM KCl, 1.4 mM KH₂PO₄, 4.3 mM Na₂HPO₄, pH 7.4) and fixed with 4% paraformaldehyde for 15 min. After blocking with 5% bovine serum albumin (BSA) in PBS-IH for 1 h, cells were incubated with mouse anti-myc 9E10 monoclonal antibody (Developmental Studies Hybridoma Bank, The University of Iowa, Iowa City, IA, USA) 1:40 diluted in PBS-IH containing 0.5% BSA for 1 h. Cells were then washed and incubated with Alexa Fluor 488-labeled goat anti-mouse antibody (Invitrogen, Grand Island, NY, USA) 1:2000 diluted in PBS-IH containing 0.5% BSA for 1 h. Cells were washed, covered with Vectashield mounting media (Vector Laboratories, Burlingame, CA, USA) and a glass coverslip, and dried overnight at 4°C. Images were taken using a Nikon A1 confocal microscope. All the steps were performed at room temperature unless mentioned otherwise.

HEK293T cells were transiently transfected with hMC4R or LmMC4R plasmid with N-terminal c-myc tag. Forty-eight hours after transfection, cells were incubated with mouse anti-myc 9E10 monoclonal antibody (Developmental Studies Hybridoma Bank, The University of Iowa, Iowa City, IA, USA) diluted 1:40 for 1 h. Cells were then washed and incubated with Alexa Fluor 488-labeled goat anti-mouse antibody (Invitrogen, Grand Island, NY, USA) diluted 1:2,000 for 1 h. The C6 Accuri Cytometer (Accuri Cytometers, Ann Arbor, MI, USA) was used for analysis. Fluorescence of cells expressing the empty vector (pcDNA3.1) was used for background staining. The expression of the LmMC4R was calculated as percentage of hMC4R expression using the following formula: [(LmMC4R – pcDNA3.1)/(hMC4R – pcDNA3.1) ×100%] (42 (link)).