Ix81 fluoview fv1000 laser scanning confocal microscope

Cells were seeded on coverslips and incubated in phenol-red-free and serum-free medium for 4 h. Cell fixation, methanol permeabilization, and staining were performed according to Cell Signaling guidelines. GR was visualized with the GR polyclonal (H300) antibody (Santa Cruz, Germany), used at 1:200, followed by probing with Alexa Fluor 488 (Invitrogen, Belgium). Nuclei were visualized using 4′,6-diamidino-2-phenylindole (DAPI) staining. A motorized inverted IX81 FluoView FV1000 laser scanning confocal microscope (Olympus) was used to record high-resolution images. Assessment of intracellular localization of protein signal was done double blind.

Cell fixation, methanol permeabilization and staining of A549 or HeLa cells were performed according to Cell Signaling guidelines. GR was visualized with the GR polyclonal (H300) antibody (Santa Cruz) while STRN3 was visualized with the STRN3 monoclonal (N20) antibody (Santa Cruz), both used at 1:200, followed by probing with Alexa Fluor® 488 (Invitrogen) and/or Alexa Fluor® 568 (Invitrogen), respectively at a concentration of 1:1000. Nuclei were visualized using 4′,6-diamidino-2-phenylindole (DAPI) staining at a concentration of 1:100. A motorized inverted IX81 FluoView FV1000 laser scanning confocal microscope (Olympus) was used to record high-resolution images.