Ix81 confocal fluorescence microscopy

Wild-type and mutant AQP0 with FLAG tag were transfected separately into HEK 293T cells. For co-localization studies, FLAG-WT-AQP0 and Myc-Y219*-AQP0 constructs were co-transfected. Immunofluorescence studies were performed as described by Varadaraj et al [20 (link)]. Briefly, 24 h after transfection, cells cultured on glass coverslips were fixed in buffer containing 4% paraformaldehyde, and then counterstained with the nucleus-staining dye, DAPI. HEK 293T cells were subjected to immunofluorescence staining using c-Myc (9E 10) mouse monoclonal IgG (Santa Cruz Biotechnology, Shanghai, China) and rabbit anti-FLAG IgG(MEDICAL & BIOLOGICAL LABORATORIES CO.,LTD., Beijing, China), followed by Dylight 594 goat anti-mouse IgG and Dylight 488 goat anti-rabbitIgG(Earthox, San Francisco, CA,USA). All samples were analyzed by Olympus IX81 confocal fluorescence microscopy. Images were digitized and merged using FV1000 Viewer (Ver.3.0a) software (Olympus). Cells expressing the pCMV-3Tag-6or pCMV-3Tag-7 expression plasmid were used as negative controls. The assay was repeated three times.

HeLa cells were cultured on glass bottom dishes. Twenty-four hours after transfection, the cells were fixed with 4% paraformaldehyde for 30 min permeabilized with 0.05% Triton-X 100 for 10 min and blocked with 5% bovine serum albumin (BSA) for 1 h. The cells were incubated with anti-alpha A crystallin antibody (Abcam) at a 1:200 dilution at 4 °C overnight, followed by Dylight 488 goat anti-rabbit IgG (Earthox, San Francisco, CA, USA) at a 1:800 dilution for one hour at room temperature. The nucleus was stained with 500 ng/ml of DAPI (Sigma Aldrich). The images were acquired with Olympus IX81 confocal fluorescence microscopy and analysed by FV1000 Viewer (Ver.3.0a) software (Olympus). The assays were repeated three times.