710 meta confocal laser scanning microscope

The K18 protein is known to dimerize with K8 protein to form K8/K18 filaments in ovarian steroidogenic cells [30 (link), 38 (link)]. KGN cells grown on coverslips were washed with PBS, then fixed with ice-cold 4 % paraformaldehyde (PFA) in PBS (pH 7.4) for 10 min. The fixed cells were permeabilized for 10 min in 0.4 % Triton-X-100 in PBS. Cells were washed and blocked with blocking buffer (0.2 % Triton-X-100 in PBS with 10 % normal donkey serum) for 30 min at room temperature. Cells were labeled with mouse anti-human K18-FITC-conjugated antibody (CY90; Sigma-Aldrich, St. Louis, MO) diluted 1:100 in blocking buffer for 3.5 h at room temperature. Cells were washed again and probed with Rhodamine Phalloidin (1:1500, Life Technologies, Grand Island, NY) and 4′,6-diamidino-2-phenylindole (DAPI; 30 nM) in blocking buffer for 30 min at room temperature. For the negative control, KGN cells were also exposed to a mouse anti- human IgG-FITC-conjugated antibody (Sigma-Aldrich, St. Louis, MO) diluted 1: 100 in blocking buffer in place of the primary. The coverslips were washed and then mounted with Fluoromount-G® (Southern Biotechnology Associates, Inc, (Birmingham, AL). Images were captured using a Zeiss 710 Meta Confocal Laser Scanning Microscope and analyzed using the Zeiss Zen 2010 software (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA).