Mouse anti acetylated α tubulin antibody

Published procedures were used for staining [30 (link), 31 ] and thick sectioning for the neural tube [32 (link)] with minor modifications. Embryos were fixed in MEMFA for either 2.5 h at room temperature or overnight at 4 °C for anti-acetylated-α-tubulin, anti-B9D1 and anti-γ-tubulin antibodies or in methanol for 48 h at 4 °C for anti-centrin antibody. Fixed embryos were dehydrated completely in methanol at −20 °C for at least several hours and rehydrated consecutively with PBS. After rinsing in PBT (0.1% Triton X-100 in PBS), embryos were incubated with 10% goat serum in PBT at room temperature for at least 1 h. Samples were incubated with mouse anti-acetylated-α-tubulin antibody (1:500, Sigma), rabbit anti-B9D1 antibody (1:50, abnova), rabbit anti-γ-tubulin antibody (1:50, abcam) and mouse anti-centrin antibody (1:50, Millipore) overnight at 4 °C. Primary antibodies were recognized with Cy2 donkey anti-mouse IgG antibody (1:500, Jackson ImmunoResearch) and Alexa Flour 648 goat anti-rabbit IgG antibody (1:500, Life Technologies), respectively. Antibodies were diluted in 10% goat serum in PBT. Images were taken by confocal microscopy.

After removal of medium and three washings in PBS, cells were fixed in 2% paraformaldehyde for 10 min. and permeabilized in 0.3% triton X‐100 in PBS for 5 min. at RT. Non‐specific binding sites were blocked by incubating cells in blocking solution containing 2% foetal bovine serum, 2% foetal bovine serum fraction V and 0.2% fish gelatine for 1 hr at RT. Samples were washed three times in PBS and then incubated for 1 hr with the primary mouse anti‐acetylated α‐tubulin antibody (1:500; Sigma‐Aldrich) or the rabbit anti‐ATP6AP2 antibody (1:500; Sigma‐Aldrich), or the mouse anti‐PDI antibody (1:500; Thermo Scientific Inc, Germany). Cells were washed again three times in PBS and incubated with the corresponding 1:500 diluted secondary antibodies (Alexa Fluor 488 chicken anti‐mouse and Alexa Fluor 596 donkey anti‐rabbit) for 1 hr at RT. Cells were finally washed three times in PBS and mounted onto glass slides for 24 hrs at 4°C using fluorescent mounting medium containing DAPI (DAKO Omnis, Hamburg, Germany). Slides were imaged using a fluorescence microscope (BZ 9000; Keyence Corp, Osaka, Japan) with a 40× or a 100× plan apochromat oil‐immersion objective. Primary cilia lengths were measured using IMAGE J software.

Following EdU incorporation and detection, juveniles were washed several times in PBS + 0.1% Triton (PBT), before being treated with block solution (PBT + 10% normal goat serum, Sigma G9023) for 45–60 minutes. Mouse anti-acetylated α-tubulin antibody (Sigma T6743) was diluted to 1:300 in block solution, and animals were incubated for 12–18 hours at 4°C. The following day, animals were washed twice in PBT, followed by four PBT washes of 20–30 minutes each. Donkey anti-mouse-546 secondary antibody (Invitrogen A21203) was diluted to 1:250 in block solution, and animals were incubated for 2–4 hours at room temperature. Following two rinses in PBT, four PBT washes of 20–30 minutes each were conducted. Finally, animals were equilibrated in 80% glycerol:PBS plus 0.125 μg/μL Hoechst 33342 (Life Technologies, H3570) overnight, before being imaged and analyzed as described below.