Rhodamine redtm x goat anti rabbit igg

Cells were seeded on coverslips and were fixed with 4% paraformaldehyde for 30min at room temperature. After permeabilization with 0.2% TX-100 (Sigma, X100) for 10min at room temperature, cells were incubated with cleaved caspase 3 antibody (ab13847), cathepsin B antibody (ab33538) or TFEB antibody (sc-48784) for 2h at room temperature. After washing with PBS, cells were incubated with Rhodamine RedTM-X goat anti-rabbit IgG (Molecular Probes, R6394) and with Hoechst 34580 (Molecular Probes, H21486) for 1h. Immunofluorescence images were viewed under Olympus FluoView1000 (FV1000; Olympus) with identical acquisition parameters for the same image session and analyzed with Olympus FLUOVIEW Ver1.7a Viewer. Cleaved caspase 3 and TFEB nuclear staining were quantified with the ImageJ software. Briefly, nucleus of cell of interest was defined using the drawing tool. Mean fluorescence of the nuclear staining was obtained by selecting the “measure” option. After deducting any background staining, the mean fluorescence of at least 50 cells from each treatment was used for statistical analysis. Data is represented as mean +/- SEM (standard error of mean).