Goat anti mouse igg2b alexa fluor 594

We cultured fibroblasts from the proband and a healthy control on coverslips maintained with 10% fetal bovine serum (FBS)/Dulbecco’s minimal essential medium (DMEM). After fibroblasts reached 90% confluence, we arrested cell growth and facilitated ciliogenesis by reducing FBS/DMEM concentration (0.5%) for 48 hours. To assess cilia length and number, we performed immunofluorescent staining with anti-γ tubulin (to mark centrosomes) and anti-acetylated tubulin (to mark cilia). Briefly, after cell fixation with pre-chilled methanol (−20°C for 10 minutes) and washing with PBS and 0.1% Triton X-100 in PBS (PBS-T), cells were stained with monoclonal IgG1 anti-γ tubulin (1:1000, Sigma, St. Louis, MO) and monoclonal IgG2b anti-acetylated tubulin (1:10,000, Sigma, St. Louis, MO) for 1 hour. After washing cells with PBS-T, cells were incubated with goat anti-mouse IgG1 Alexa Fluor 488 and goat anti-mouse IgG2b Alexa Fluor 594 (Invitrogen, Carlsbad, CA) for 1 hour. Images were captured with a Nikon Eclipse Ti-E inverted microscope and a wide-field immunofluorescence microscope. At least 100 cells were scored for the presence or absence of cilia. We used Chi square testing to compare the percentage of ciliated cells. We used Image J (https://imagej.net) to measure cilia length and Student’s t-test to compare cilia length in the proband and control fibroblasts.