Computational protocol: Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a-/- mutants

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Protocol publication

[…] Embryos at 27 hpf were fixed at 4°C overnight in 4% PFA, then dechorionated, dehydrated in methanol and stored at -20°C. Prior to immunostaining embryos were rehydrated in PBS, permeabilised for 5 min in proteinase K (20 μg/ml) and post-fixed in 4% PFA for 30 min. Larvae were treated with blocking solution (2% normal goat serum, 1% Triton X-100, 1% Tween-20 in PBS) for 60 min followed by incubation in the primary antibody: anti-phospho-histone H3 at 1:200 dilution (Upstate Biotech) for 20 hours. Primary antibody binding was detected with Cy3-conjugated goat anti-rabbit secondary antibody (dilution 1:200). Nuclei were counter stained with DAPI. Z-stack images of whole larval eyes (optical sections at 2 μm interval) were taken with Zeiss LMS 510 Meta laser confocal microscope (with 63 × objective). Images were analysed with Imaris v7.2.3 software. To quantify the proportion of mitotic cells, embryos at 27 hpf were fixed in PFA, cryoprotected and sectioned (12 μm thickness). Mitotic nuclei were labelled with anti-phospho-histone H3 antibody (Upstate Biotech) and counter stained with DAPI. Images of sections through the eyes, head and spinal cord (at least 3 per animal) were taken with Zeiss AxioImager M1 microscope (objective 100×) and all PH3 positive and DAPI labelled nuclei from the field (89.5 μm × 67.1 μm) were counted. The percentage of mitotic cells in 5 mutant and 5 wild type larvae was compared. [...] Protein samples were separated according to their apparent molecular mass under denaturing conditions on NuPAGE 4-12% Bis-Tris Gel, 1.0 mm × 17 well gels (Invitrogen), and transferred to PVDF membrane. After blocking in 5% Bovine Serum Albumin (BSA) in PBS-0.1% Tween-20, membranes were incubated with either anti-phospho-Histone H3 (Ser10) antibody from Millipore to detect mitosis (Cat. # 06-570) or GAPDH antibody from Cell Signaling (#3900), followed by incubation with horse-radish peroxidase-conjugated secondary antibodies. Proteins were visualized using Enhanced Chemiluminescence Western Blotting Substrate (Pierce; #32106). Densitometry was performed using the ImageJ program http://rsbweb.nih.gov/ij/. […]

Pipeline specifications

Software tools Imaris, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Danio rerio, Homo sapiens, Mus musculus
Diseases Neoplasms