Axioimager m1 compound microscope

Whole-embryo RNA in situ hybridization analysis of spaw was performed as previously described (Wang et al., 2011 (link)). For immunostaining, embryos were fixed overnight at 4° C in 4% paraformaldehyde in PBS with 0.5% Triton X-100 and then processed for whole mount antibody staining as described (Tay et al., 2013 (link)). Primary antibodies were: anti-aPKC (Santa Cruz sc-216) (1:200 dilution) and anti-acetylated tubulin (Sigma T6793) (1:400). AlexaFluor 488 and 568 (anti mouse and anti-rabbit) fluorescent secondary antibodies (Invitrogen) were used at 1:200 dilutions. Embryos were imaged using a Zeiss AxioImager M1 compound microscope or a Perkin-Elmer Ultra View Vox spinning disk confocal microscope. When necessary, images were rotated for uniform orientations with anterior at top.

Whole mount RNA in situ hybridizations were performed as previously described (Wang et al., 2011 (link)). For immunostaining, embryos were fixed overnight at 4° C in 4% paraformaldehyde, sucrose buffer (4% sucrose, 0.1 M NaPO₄, 0.3 M CaCl₂) and 0.5% Triton X-100 and then processed for whole mount antibody staining as described (Tay et al., 2013 (link)). Primary antibodies: anti-aPKC (Santa Cruz sc-216) (1:200 dilution), anti-acetylated tubulin (Sigma T6793) (1:400), anti-GFP (Invitrogen A11120) (1:400), anti-phosphorylated Histone H3 (Santa Cruz sc8656R) (1:400) and anti-ATP6AP1 (Sigma A1486) (1:200). For Atp6v1f immunostaining, 4% paraformaldehyde + 0.02% Tween was used to fix embryos and the primary antibody was anti-Atp6v1a (Proteintech Group 17115-1-AP) (1:125) as described (Einhorn et al., 2012 (link)). A second Atp6v1a antibody (GenScript JP_A00938-40)(1:100) was used to verify staining. AlexaFluor 488, 568 and 647 fluorescent secondary antibodies (Invitrogen) were used at 1:200 dilutions. DAPI (Sigma) (1:500) was used to stain nuclei. Embryos were imaged using a Zeiss AxioImager M1 compound microscope or a Perkin-Elmer Ultra View Vox spinning disk confocal microscope. When necessary, images were rotated for uniform orientations.