Embryonic Rat Thoracic Aorta Medial Layer Myoblast Cells (A-10 Line)
In a double immunofluorescence experiment, the adherent monolayer culture of rat thoracic aorta cells illustrated above was fixed, permeabilized, blocked with 10 percent normal goat serum, and treated with a cocktail of mouse anti-PDI (protein disulfide isomerase) and rabbit anti-PMP 70 (peroxisomal membrane protein) primary antibodies, followed by goat anti-mouse and anti-rabbit secondary antibodies (IgG) conjugated to Alexa Fluor 568 (red fluorescence) and Alexa Fluor 750, respectively. The filamentous actin network was counterstained with Alexa Fluor 488 (green fluorescence) conjugated to phalloidin, mitochondria were labeled with MitoTracker Deep Red 633, and nuclei were targeted with Hoechst 33258 (cyan fluorescence). Images were recorded in grayscale with a 12-bit digital camera coupled to either a Nikon E-600 or Eclipse 80i microscope equipped with bandpass emission fluorescence filter optical blocks. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles with the exception of MitoTracker Deep Red 633, which was pseudocolored purple, and Alexa Fluor 750, which was pseudo colored yellow.