Alexa fluor 555 conjugated goat anti rabbit igg antibody

Immunofluorescence assays were conducted as described previously 24 (link) with some modifications: primary antibodies were a rabbit polyclonal anti-pendrin antibody, that recognizes the amino acids CKDPLDLMEAEMNAEELDVQDEAMRRLAS (1:500; provided by Prof. Min Goo Lee at Yonsei University College of Medicine, Seoul, Korea), and a mouse anti-Kcnj10 antibody (1:500; Abnova, Taipei, Taiwan) diluted in the blocking solution, and the secondary antibody was an Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody (1:1000; Invitrogen, La Jolla, CA, USA) diluted in the blocking solution. The slides were mounted with an aqueous medium (Fluoromount, Sigma-Aldrich, St. Louis, MO, USA). GFP fluorescence was detected directly. Images were captured by conventional microcopy (Axioscope, Axio Imager.A2, Carl Zeiss, Inc., Oberkochen, Germany) or confocal laser microscopy (LSM700 with ZEN 2012 software, Carl Zeiss, Inc., Oberkochen, Germany). DAPI and pendrin expressing epithelial cell in the endolymphatic sac were counted using a computer algorithm (ImageJ, National Institutes of Health, Bethesda, MD, USA). Counts were verified by manual counting. The density of pendrin expressing epithelial cells in the endolymphatic sac was determined as ratio of pendrin- and DAPI-positive cells.