Mouse anti acetylated α tubulin 6 11b 1

Embryos were fixed for 2 h at room temperature in 4% paraformaldehyde in PBS (PFA). Embryos were stored in methanol, and then rehydrated in a gradient of PBS/methanol. Embryos were permeabilized in acetone, blocked in PBDT (PBS, 1% BSA, 1% DMSO and 1% Triton X-100) plus 5% sheep serum. Primary and secondary antibodies were incubated and washed in PBDT.
Primary antibodies used include mouse anti-acetylated-α-tubulin 6-11B-1 (1:500 for zebrafish embryos, Sigma, #T6793), mouse anti-γ-tubulin GTU-88 (1:500, Sigma, #T6557), rabbit anti-GFP (1:500, Abcam, #ab6556; Torrey Pines Biolabs, #TP401), rabbit anti-Arl13b (Duldulao et al., 2009 (link)) (1:200), DAPI (4′,6-diamidino-2-phenylindole; Molecular Probes, #D1306) was used to label cell nuclei. Alexa fluorophore-labeled anti-rabbit or anti-mouse secondary antibodies (Molecular Probes) were used to visualize staining.

Rabbit anti-CFAP70 (HPA037582) and mouse anti-acetylated α-tubulin (6-11B-1) were from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-IFT88 (13967-1-AP) was from Proteintech (Rosemont, IL, USA). Mouse anti-dynein intermediate chain (DNAIC1, GTX26304) was from GeneTex (Irvine, CA, USA). Rabbit anti-green fluorescent protein (GFP) (A-6455) and Alexa-Fluor-conjugated secondary antibodies were from Life Technologies (Carlsbad, CA, USA). Horseradish-peroxidase- and alkaline-phosphatase-conjugated secondary antibodies were from Cell Signaling Technologies (Danvers, MA, USA).
Rabbit polyclonal antibodies against Chlamydomonas reinhardtii FAP70 were produced using a synthetic oligopeptide SEGGVYQYSVVKHFC (Cosmo Bio, Tokyo, Japan) at EveBioscience (Wakayama, Japan). For affinity purification of anti-FAP70 antibodies, a GeneArt Strings DNA fragment corresponding to the coding sequence of FAP70 in exons 2 and 3 was synthesized by Thermo Fisher Scientific (Waltham, MA, USA) and ligated into pGEX6P-1 (GE Healthcare Japan, Tokyo, Japan). The purified recombinant FAP70 fragment was resolved by SDS-PAGE, transferred onto a PVDF membrane, and the membrane strip was used for affinity purification of the antibodies as described previously [26 (link),27 (link)].