Immunohistochemical analysis and subcellular localization of RYR1 and of the α 1 subunit of the DHPR in single muscle fibers from WT and ryr3−/− mice. (A) Left and central panels show the staining obtained using rabbit anti-RYR1 mAb D4E1 (green in merged image) and mouse anti-Cav1.1 (red in merged image), respectively. The panel on the right shows the merged images as well as location of the myonuclei (DAPI, blue). (B) Left and central panels show the staining obtained using mouse anti-Cav1.1 (red in merged image) and rabbit anti-Cav1.2 (green in merged image), respectively. The panel on the right shows the merged images as well as location of the myonuclei (DAPI, blue). The bottom panels show staining of mouse EDL fibers, which are negative for Cav1.2 and were used as staining control. All images in B were acquired using the same settings for the laser intensities and acquisition parameters. Images were acquired using a Nikon A1 plus confocal microscope equipped with a Plan Apo 60× oil objective (numerical aperture, 1.4) and stained as described in Materials and methods. Orange pixels show areas of colocalization. Scale bars, 30 µm.
