C2 confocal microscopy system

Immunofluorescence analysis was performed as described previously [21 (link)]. Images were acquired by using the confocal microscope PCM Eclipse TE300 or the C2 Confocal Microscopy System (both from Nikon Instruments, Tokyo, Japan). Merged images were obtained with EZ2000 or NIS Element software. Quantification of colocalization, expressed in terms of overlap coefficient (R), was calculated on several randomly selected cells from different slides by using the WCIF ImageJ software.

Fibroblasts from one patient homozygous for TMEM17 mutations and from his healthy mother (heterozygous carrier) were plated on coverslips and cultured in DMEM with 20% FBS until they were 80% confluent after being starved for 24 h in DMEM 0.1% FBS to allow cilia formation. Cells were fixed in cold methanol, and coverslips were rinsed and blocked in PBS with 10% BSA prior to incubation with antibodies. Fixed cells were incubated with the following antibodies: acetylated-α-tubulin (1:1000, SIGMA), γ-tubulin (1:500, SIGMA) overnight followed by incubation with goat anti-mouse Alexa fluor 555 (1:3000), and goat anti-rabbit Alexa fluor 488 (1:3000). Nuclei were stained with Hoechst (Invitrogen). Images were captured with a confocal microscopy (C2 Confocal Microscopy System), by using the laser lines 488 nm (green) or the 561 nm (red) and a 60X 1.4 NA Plan Apo objective (Nikon Corporation) controlled by NIS Element AR 4.13.04 software. To count cilia, acetylated-α-tubulin and γ-tubulin positive cilia were manually counted within 15 images for each phenotype in at least three independent experiments. Variables were analysed by Student’s t test, and a value of p < 0.05 was deemed statistically significant. Values are expressed as standard error (S.E.).