We provide an expert step-by-step protocol with adapted conditions for handling procedures (tsA-201 cell culture, transient transfection, incubation time and temperature at 28 °C or 37 °C and immunostaining) to address the L-type calcium-channel pore Cav1.2 in an immunofluorescent approach.
We performed immunocytochemical analysis of Cav1.2 expression at single-cell level in combination with detection of different markers for cellular organelles. We show confluency levels and shapes of tsA-201 cells at different time points during an experiment. Our experiments reveal sufficient levels of Cav1.2 protein and a correct Cav1.2 expression pattern in polygonal shaped cells already 12 h after transfection.
A sequence of elaborated protocol modifications allows subcellular localization analysis of Cav1.2 in an immunocytochemical approach. We provide a protocol that may be used to achieve insights into physiological and pathophysiological processes involving voltage gated calcium channels. Our protocol may be used for expression analysis of other challenging proteins and efficient overexpression may be exploited in related biochemical techniques requiring immunolabels.