Immunofluorescence Analysis of IFT80 in Mouse Tissues

Immunofluorescence staining analysis of IFT80 expression in mouse tissues was performed as described previously [12 (link)]. The cells were seeded at 4 × 10⁴ cells/well on 24-well plates, and incubated overnight, followed by infection with the IFT80 and/or control plasmid for 24 h. The cells were sequentially induced with media containing 10% FBS for 3 days. After 48 h of serum starvation (without FBS), the cells were fixed with 100% ice-cold methanol for 10 min, and washed 3 times with PBS. Fixed cells were blocked with 5% BSA in PBS for 60 min. The cells were then incubated with primary antibody diluted in PBS containing 1% BSA for 1 h. The primary antibodies used were as follows: anti-IFT80 antibody (Abnova, Walnut, CA, USA; 1:500) and mouse monoclonal anti-acetylated tubulin antibody (Sigma, T7451, 6-11B-1, 1:1,000). After washing 3 times with PBS, Alexa Fluor 488- and Alexa Fluor 568 (Invitrogen, Grand Island, NY, USA)-conjugated anti-rabbit or anti-mouse IgG was added in PBS with 1% BSA for 1 h. In the final washes, 6-diamidino-2-phenylindole (DAPI) (Sigma) was added, and used as a counterstain for nuclei. Fluorescence images were acquired using a Zeiss Axio Imager microscope.