Cryostar nk70

Embryos from a sgo1^+/− incross were fixed at 3 dpf in 4% paraformaldehyde and genotyped by tailfin clips to separate homozygous mutant from wild‐type larvae. Three washes of 10 min were performed using 4% sucrose (in phosphate buffer) followed by an 30 minute incubation at room temperature in 30% sucrose (in phosphate buffer). Larvae were embedded in tissue‐freezing medium (Leica, Lot# 03811456), frozen on dry ice, and kept at −80°C. Cryo‐sectioning using Cryostar NK70 (Thermo Scientific, Breda, The Netherlands) was performed to obtain 7 μm thin sections. Staining of sgo1^−/− and wild‐type larvae was performed. Using Roche In Situ Cell Detection Kit (TMR red# 12156792910). Sections were rinsed in PBT (PBS +0.1% Tween 20), incubated with collagenase Type II (17101015, Gibco) for 30 minutes at room temperature, then blocked for 1 hour at RT with PBT supplied with DMSO and FBS, incubation with Anti‐phospho Histone H3 (Ser10) at 1:100 (06‐570, Millipore) was performed overnight at 4°C. Slides were washed multiple times in PBT and incubated in labeling solution from the kit, supplied with the secondary antibody Alexa 555 Anti‐rabbit (1:500) (Cat# A‐21428, Invitrogen) and DAPI (1:1000) for 1 hour at RT, washed multiple times in PBT and coverslip was added. Imaging was performed using Olympus Slideview VS200 digital slide scanner.

For paraffin sections, adult zebrafish hearts were dissected and fixed in 4% paraformaldehyde (dissolved in phosphate buffer containing 4% sucrose) at 4 °C overnight, washed twice in PBS, dehydrated in EtOH, and embedded in paraffin. Serial sections were made using a microtome (Leica RM2035, Leica, Wetzlar, Germany). Different section thicknesses were used, depending on the size/age of the sample: 10 μm for adult hearts, 8 μm for juvenile hearts, and 6 μm for embryos. For cryosections, zebrafish hearts were extracted and fixed in 4% paraformaldehyde (in phosphate buffer) for 4 h at room temperature (RT). Three washes of 30 min were performed using 4% sucrose (in phosphate buffer) followed by an overnight incubation at 4 °C in 30% sucrose (in phosphate buffer). Hearts were embedded in tissue-freezing medium (Leica, Lot# 03811456), frozen on dry ice, and kept at −80 °C. Cryo-sectioning using Cryostar NK70 (Thermo Scientific, Breda, The Netherlands) was performed to obtain 10 μm thin sections. Images of extracted whole hearts were acquired using a Leica M165 FC stereomicroscope.

For paraffin sections, adult zebrafish hearts were dissected and fixed in 4% paraformaldehyde (dissolved in phosphate buffer containing 4% sucrose) at 4 °C overnight, washed twice in PBS, dehydrated in EtOH, and embedded in paraffin. Serial sections were made at 10 µm using a microtome (Leica, RM2035). For cryosections, zebrafish hearts were extracted and fixed in 4% paraformaldehyde (in phosphate buffer) for 4 h at room temperature (RT). Three washes of 30 min were performed using 4% sucrose (in phosphate buffer) followed by overnight incubation at 4 °C in 30% sucrose (in phosphate buffer). Hearts were embedded in tissue freezing medium (Leica, Lot# 03811456), frozen on dry ice and kept at −80 °C. Cryo-sectioning using Cryostar NK70 (Thermo Scientific) was performed to obtain 10 µm thin sections. Images of extracted whole hearts were acquired using a Leica M165 FC stereo microscope.