Mouse anti acetylated tubulin t6793

For whole mount and cryosection immunostaining, embryos were fixed in 4% paraformaldehyde (PFA) at 4°C overnight at different stages (between 11 to 24 hpf). Embryos were blocked in 10% normal goat serum (Sigma-Aldrich, St Louis, MO, USA) for 2 hours at room temperature. The following primary antibodies were used in this study: mouse-anti-ZO-1 (339111; Zymed Laboratories, South San Francisco, CA, USA) at 1:300; rabbit-anti-aPKC (C-20; Santa Cruz Biotechnology, Dallas, TX, USA) at 1:500; rabbit-anti-GFAP (Z0334; DakoCytomation, Glostrup, Denmark) at 1:500; mouse-anti-MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) at 1:50; mouse-anti-acetylated-tubulin (T6793; Sigma-Aldrich); and rabbit-anti-phospho-histone H3 (Upstate Biotechnology, Lake Placid, NY, USA) at 1:200, diluted in 2.5% normal goat serum (Sigma-Aldrich). For secondary antibodies, anti-rabbit and anti-mouse Alexa 488, Alexa 568 and Alexa 633 (Molecular Probes, Eugene, OR, USA) were used at 1:800 in 2.5% normal goat serum. Sections were cut every 14 to 16 μm on a Leica 5100 or Cryo-Star HM 560 MV Micron microtomes.

Fixed embryos were serially rehydrated from 100% methanol into PBST [0.2% Tween-20 (Sigma P2287) in 1× PBS (Oxoid BR0014G)], washed three times in 0.2% Triton-X (Sigma T8787) in 1× PBS (PBSTx) and then incubated in blocking buffer for 1 h at room temperature [5% sheep serum (Gibco, 16070-096), 10 mg/ml bovine serum albumin (A2153, Sigma) and 1% DMSO (D4540, Sigma) in PBSTx] for 1 h at room temperature before an overnight incubation at 4°C with gentle agitation with the following primary antibodies: mouse anti-acetylated tubulin (T6793, Sigma, 1:500) and rabbit anti-PKC ζ (aPKC) (sc-216, Santa Cruz, 1:400) in blocking buffer (Amack et al., 2007 (link)). The next day, embryos were rinsed extensively in PBSTx, before a further overnight incubation at 4°C with gentle agitation with the following secondaries: Alexa488-conjugated donkey anti-mouse (Invitrogen A21202, 1:200) and Alexa647-conjugated donkey anti-rabbit (Invitrogen A31573, 1:200) in blocking buffer. Embryos were washed extensively on day three before dissection or embedding. Representative images of Kupffer's vesicle were taken using a Nikon A1 inverted confocal using a 40× objective.

Third-instar larvae were dissected in ice-cold PBS and fixed in 4% paraformaldehyde for 30 min. The fixed tissues were stained following standard procedures. The primary antibodies used were: mouse anti-DLG, anti-Futsch and anti-β-tubulin antibodies from the Developmental Studies Hybridoma Bank; rabbit anti-DVGlut (Daniels et al., 2004 (link)), rabbit anti-GFP (A11122, Invitrogen) at 1:1000, rabbit anti-mCherry (632496, Clontech) at 1:1000, mouse anti-acetylated tubulin (T6793, Sigma) 1:1000, mouse anti-Tau (12-6400, Invitrogen) at 1:1000, Cy3-conjugated goat anti-HRP, and Cy5-conjugated goat anti-HRP. The following secondary antibodies (from Jackson ImmunoResearch) were used: Cy3-conjugated goat anti-rabbit IgG at 1:1000, Dylight-488-conjugated anti-mouse IgG at 1:1000, and Alexa-Fluor-647-conjugated goat anti-HRP at 1:1000.