Mouse monoclonal anti acetylated alpha tubulin

Patient and control fibroblasts were prepared as described previously. Ciliary axoneme and IFT22, IFT25, IFT46, IFT88, GLI1, GLI2 or SMO were stained overnight at 4°C using mouse monoclonal anti-acetylated alpha-tubulin (1:1000, Sigma Aldrich), rabbit polyclonal anti-IFT22, (1:200, Sigma Aldrich), rabbit polyclonal anti-IFT25 (1:100, Thermo Scientific); rabbit poly/monoclonal anti-IFT46 (provided by Frédéric Mallein-Gerin); rabbit poly/monclonal anti-IFT88 (provided by Chantal Desdouets); rabbit polyclonal anti-GLI1 (1:50, Abcam), goat polyclonal anti-GLI2N, (1:50, Santa Cruz) or rabbit polyclonal anti-Smoothened (1:100, Abcam) antibodies. Secondary antibodies were used as described above. Fluorescent images were obtained with a Zeiss LSM700 (Carl Zeiss SAS) laser scanning microscope.

Patient and control fibroblasts were cultured and prepared as described previously. IFT81 was stained overnight at 4 °C with, centrioles, ciliary axoneme or subdistal appendages using rabbit polyclonal anti-IFT81 (1:200, Proteintech), goat monoclonal gamma-tubulin (1:200; Santa Cruz), mouse monoclonal anti-acetylated alpha-tubulin (1:1000; Sigma Aldrich) or mouse monoclonal anti-ODF2 (1:100, Novus) antibodies. Secondary antibodies were used as described above. Images were recorded from a Gated STED Leica SP8. The intensity of IFT81 staining at the cilia base and at the tip was quantified by using imageJ software. Average fluorescent intensities were determined from the region of interest drawn around the cilium from maximum intensity Z projection images. Centriolar and tip intensites and ratio of the one to the other were compared in patient and control cell lines.

Serum-starved primary cultured fibroblasts from four human controls and patient (NCK033) were fixed with methanol 100% and blocked with BSA 3% and triton 0.1 % in PBS. Ciliary axoneme and basal bodies were stained overnight at 4°C using mouse monoclonal anti-acetylated alpha-tubulin (Sigma Aldrich; 1:1000) and rabbit polyclonal anti-pericentrin (1:1000, Abcam) antibodies, respectively. Primary antibodies were labeled for 1 hour at room temperature using AlexaFluor 594 goat anti-mouse (Molecular probe; 1:1000,) and AlexaFluor 488 goat anti-rabbit (1:1000 Invitrogene) secondary antibodies. Images were recorded from Zeiss LSM700 microscope (×40 magnification, Carl Zeiss). Mean numbers of ciliated cells were calculated from 973 patient cells and 895 cells from four individual controls, in two independent experiments. Cilia lengths were measured from the same immunofluorescent images. Mean numbers of cilia <3 μm and >3μm in length were determined from 100 and 150 patient and control cells, respectively. Data from patient and control cells were compared using the PLSD of Fischer according to the significance of the Student’s test.