Anti histone h3 phospho s10

Avian embryos were fixed with 4% formaldehyde, embedded in paraffin wax and sectioned at 5 or 8 μm. Immunostaining for GFP (Molecular Probes), Islet-1, and Brdu (all from DSHB) was as described [55 (link)]. HNK-1 (mouse anti-human CD57) was from BD Biosciences. Anti-Histone H3 (phospho S10) was from Abcam (mAbcam 14,955). Nuclei were visualized with Hoechst.
For YAP immunostaining, a rabbit polyclonal (Santa Cruz SC-15407, YAP H-125) against aa206–330 of the human YAP and mouse anti-YAP [Santa Cruz SC-101199; YAP (63.7)] were used and yielded similar results. Detection of DNA fragmentation was done by TUNEL (ab66110, Abcam) according to manufacturer’s instructions.
In situ hybridization for Foxd3 [17 (link)] and EdnrB2 [56 (link)] was performed as described [57 (link)].
Sections were photographed using a DP73 (Olympus) cooled CCD digital camera mounted on a BX51 microscope (Olympus). For figure preparation, images were exported into Photoshop CS2 (Adobe). If necessary, the brightness and contrast were adjusted to the entire image. In all transverse sections presented, lateral is to the left and dorsal is top.

A modified version of the Aurora A mislocalisation assay using the same cells, doxycycline-treated controls, drugging plan, Bortezomib treatment, fixing protocol, Hoechst staining and secondary antibodies as above. The primary antibodies used were anti-phospho Aurora A Thr288 (Cell Signaling, #3079, 1:500) and anti-Histone H3 phospho S10 (Abcam, 14955, 1:2000). High-content imaging settings were the same as for the mislocalisation assay, but analysis was performed using the CellHealthProfiling v4 Bioapplication. Cells were identified in the Hoechst channel as above, and the resulting nuclear mask used for all subsequent measurements. Mitotic cells were selected from the images on the basis of phospho-histone H3 positivity, using a threshold 10 times above the image background. These selected cells were then measured for phospho-Aurora A staining intensity within a target area defined by expanding the nuclear mask by 8 pixels then collapsing it using a threshold for the phospho-Aurora channel, set to 3 times the background staining in this channel. Assay thresholds were calculated from the negative control wells of each plate as for the mislocalisation assay and ‘% dephosphorylated Aurora A T288’ scores were calculated for each well. EC₅₀ values were calculated from four-parameter dose-response curves that were fitted using Prism GraphPad software (La Jolla, CA).

Guts were fixed for 1 h in a 4% PFA in PBS solution, washed in PBS, then let overnight in 30% sucrose in water solutions, and embedded the next day in OCT compound (VWR) on dry ice. Thin (14 μm) slices were cut at −20 °C in a Leica cryotome and deposited on Thermofrost glass slides. After rehydration, the slides were blocked for 15 min in a 1% BSA and 0.1% triton in PBS solution, the slides were then incubated overnight in anti-α smooth muscle actin antibody (Abcam, ref5694, dilution 1:2000), anti βIII-tubulin antibody (Abcam, ref14545, dilution 1:1000) or anti-Histone H3 (phospho S10) (Abcam, ref14955, dilution 1:200) solution composed of 1% BSA in PBS; the following day, after washing, CY3- and A488-conjugated secondary antibodies (ThermoFisher, dilution 1:400 in PBS) were applied for 2 h. The slides were washed, sealed with a coverslip and immediately imaged with an epifluorescence or confocal microscope. Epithelial cells could be visualized in brightfield.