Mouse monoclonal anti mhc

Cells seeded at density of 2.5 × 10³ per cm² in Lab-Tek II 8-well chamber slides (Nalge Nunc, Rochester, NY, USA) were fixed in 4% formaldehyde for 10 min at room temperature. After washing cells in PBS three times, cells were subsequently incubated with ice-cold methanol for 20 min at −20 °C. The following primary antibodies diluted 1:200 including mouse monoclonal anti-α-SMA and mouse monoclonal anti-MHC (Santa Cruz Biotechnology, Aarhus, Denmark) were applied. After reaction with primary antibodies at 4 °C overnight, the cells were washed with PBS three times. Alexa 488-conjugated goat anti-mouse secondary antibody (Life Technologies Europe, Naerum, Denmark) was used to detect the localization of different antibodies respectively, and cell nuclei were counterstained with Hoechst 33342. The images were acquired and processed using the microscope Axio Observer Z1 (Carl Zeiss, Göttingen, Germany).

The following primary antibodies were used: rabbit polyclonal anti-MURC/cavin-4 for immunohistochemical analysis (Code: HPA020973, Sigma-Aldrich, Milan, Italy); goat polyclonal anti-MURC/cavin-4 for immunoblotting analysis (Code: SC-163021, Santa Cruz Biotechnology, Dallas, TX, USA); rabbit polyclonal anti-MURC/cavin-4 for immunofluorescence analysis (as described in [52 (link)]); mouse monoclonal anti-Cav-3 (Code: 610420, BD Transduction Laboratories, Buccinasco, Milan, Italy); rabbit anti-myogenin (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500 dilution); mouse monoclonal anti-MHC (Code: SC-32732, Santa Cruz Biotechnology, Dallas, TX, USA); mouse monoclonal anti-total and—phosphorylated ERK1/2 (extracellular regulated kinase 1/2) (Tyr204) (Code: SC-135900 and SC-7383, Santa Cruz Biotechnology, Dallas, TX, USA); mouse monoclonal anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (Code: MAB374, Millipore, Darmstadt, Germany) and mouse monoclonal anti-alpha-tubulin (Code: T5168, Sigma-Aldrich, Milan, Italy).