Biotinylated swine anti rabbit immunoglobulin

Cells grown on coverslips were fixed with 4 % paraformaldehyde for 15 min, permeabilized with 0.1 % Na-citrate, 0.1 % Triton X-100, pH 7 for 5 min and, where indicated, counterstained with 4′,6′-diamidino-2-penylindole (DAPI, Sigma-Aldrich, Brøndby, Denmark). Actin filaments where visualized by Alexa Fluor 488 phalloidin staining (Invitrogen) for 20 min at room temperature according to the manufacturer’s recommendations. Localization of NF-κB was performed by incubating the cells with rabbit monoclonal anti-NF-κB/p65/RelA antibody (Cell Signaling Technology, Herlev, Denmark) at 4 °C overnight followed by labeling with biotinylated swine anti-rabbit immunoglobulin (Dako, Glostrup, Denmark) for 1 h at room temperature and Alexa Fluor 488-conjugated streptavidin (Life Technologies) for 30 min at room temperature. Cells were analyzed for HSP90-CDC37 interaction by in situ proximity ligation assay (PLA, Olink Biosciences, Uppsala, Sweden) according to the manufacturer’s recommendations. The number of positive signals was determined using Blobfinder software (http://www.cb.uu.se/~amin/BlobFinder). Cells were analyzed on a Leica DMRBE microscope equipped with a DFC 420C camera and Leica Application Suite V 3.3.0 software (Leica Microsystem, Wetzler, Germany).

Immunostaining was carried out essentially as described in [21 (link)] employing rabbit monoclonal anti-HSF1 antibody (Cell Signaling Technology) followed by incubation with biotinylated swine anti-rabbit immunoglobulin (Dako, Glostrup, Denmark) and streptavidin-conjugated Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MA, USA). Cells were counterstained with 4’,6’-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and analyzed on a Leica DMRBE microscope equipped with a DFC 420C camera and Leica Application Suite V 3.3.0 software (Leica Microsystem, Wetzler, Germany) at 400x magnification.