Spot 2 digital camera

Following various treatments, podocytes in culture were fixed in 4% paraformaldehyde in PBS and permeabilized filamentous actin (F-actin) was stained by incubation at 4°C with rhodamine conjugated phalloidin (Cytoskeleton Inc., Denver, CO). Then, cells were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) and mounted with Vectashield mounting medium. Images were documented using an Olympus fluorescence microscope equipped with a Spot II digital camera. The percentage of F-actin or stress fibers under fluorescent microscopy were analyzed and quantitated as described elsewhere [56 (link)].

Cultured cells or frozen kidney cryostat sections were fixed and processed for fluorescent staining. For dual color fluorescent immunohistochemistry staining, samples were stained with primary antibodies and followed by applying the Alexa Fluor-conjugated secondary antibodies (Invitrogen). As a negative control, the primary antibodies were replaced by preimmune IgG from the same species; no staining occurred. Finally, samples were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). For fluorescence microscopy, all sections were analyzed at the same time to exclude artifacts due to variable decay of the fluorochrome. Sections were examined using an Olympus fluorescence microscope equipped with a Spot II digital camera. For dual-color staining, images were acquired sequentially to avoid dye interference. ImageJ software was used for post processing of the images, e.g. scaling, merging, and colocalization analysis.