Phospho histone h3

Fixation and staining of embryos was carried out as described previously [36 (link)]. The following antibodies were used at a 1/1000 dilution: DM1A (Sigma), phospho-histone H3 (Abcam), ZYG-1 [19 (link)], SPD-2 [50 (link)] and anti-SAS-4 [53 (link)]. For live and fixed imaging we used a spinning disk confocal microscope which has been described previously [61 (link)]. To determine whether PP1 affects translation of the ZYG-1 transcript we shifted worms carrying the reporter construct to 25°C as L4s and imaged embryos the next day. Intensities of chromatin GFP at first metaphase were measured using Metamorph. Levels of ZYG-1 or SPD-2 at the centrosome were determined by quantification of average pixel intensity at the centrosome. Maximal projections of the centrosome were used for quantification of fluorescence in ImageJ 1.40g and background fluorescence was subtracted. Centrosome fluorescence was normalized to controls such that control intensity is 1. For structured illumination microscopy, embryos were immuno-labeled as usual and mounted in Vectashield (Vector Laboratories, Inc.). Samples were imaged with a DeltaVision OMX4 SIM Imaging System (Applied Precision).

For immunoblotting, proteins were extracted by lysing cells in ice-cold radioimmunoprecipitation buffer supplemented with protease and phosphatase inhibitors (Roche, Nutley, NJ, USA). Protein was quantified using the Bradford assay (Bio-Rad, Hercules, CA, USA) and 30 μg of total protein was resolved by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% skim milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 1 h, washed with TBST and incubated with antibodies against: PLK1 (Cell Signalling, Danvers, MA, USA), CDK1 (Abcam, Cambridge, UK), cyclin B1 (Abcam), phospho-Histone H3 (phospho-Ser10, Abcam), β-actin (Sigma-Aldrich) and FOXC2 (kindly provided by Dr. Naoyuki Miura, Hamamatsu University School of Medicine, Hamamatsu, Japan). For quantifications, the levels of protein was measured by ImageJ and band intensities were normalized to corresponding loading controls.

Immunohistochemistry was performed as previously described [19 (link), 40 (link)–42 (link)]. β-galactosidase (Aves BGL-1010; 1:200 dilution), Mlc2v (Synaptic Systems 310 111; 1:500), PECAM (BD Pharmingen 550274; 1:200), Phospho-Histone H3 (Abcam 4797; 1:500), and Mki67 (Dako; 1:500) antibodies were used with biotinylated secondary antibodies and streptavidin-conjugated DyLight 488 or 594 fluorophores (ThermoFisher). Images were collected on a Leica DM5000 B microscope and Leica Application Suite software.
Cell proliferation was assayed via counting of Phospho-Histone H3 positive nuclei. Left and right ventricles from E12.5 and E14.5 immunostained images were manually isolated in Adobe Photoshop, and nuclei were manually counted using Image J software. For E12.5, n≥9 sections, and for E14.5, n≥10 sections per heart were counted. Total samples analyzed were as follows: E12.5, control embryos (Hand1^LV-Cre(-); R26R^lacZ/DTA)–n = 2, RV, 35057 cells, total, LV, 35250 cells, total; LV-ablated embryos (Hand1^LV-Cre(+); R26R^lacZ/DTA)–n = 5, RV, 81471 cells, total, LV, 67618 cells, total; E14.5, control embryos (Hand1^LV-Cre(-); R26R^lacZ/DTA or Hand1^LV-Cre(+); R26R^lacZ/+)–n = 3, RV, 76254 cells, total, LV, 85121 cells, total; LV-ablated embryos–n = 4, RV, 167897 cells, total, LV, 80894 cells, total.

Mandibles from 2- to 3-month-old heterozygous and homozygous Enam^p.S55I mutant and wild-type mice (n = 4 in each category) were dissected following cervical dislocation and fixed in 4% paraformaldehyde in PBS, pH 7.4, at room temperature for 48 h. Following fixation, the mandibles were decalcified in 0.5 M EDTA, dehydrated through a graded ethanol series, cleared in chloroform, embedded as hemi-mandibles in paraffin wax, sectioned and stained with haematoxylin and eosin. Sections were examined using a DMRB microscope (Leica) with SpotTM digital camera and associated software (RTKE/SE Diagnostic Instruments Inc.). Immunofluorescence analysis was performed on mandibles prepared as above using antibodies raised against activated caspase 3 (Abcam, Cambridge, UK), amelogenin FL191, (Santa Cruz Biotechnology, Santa Cruz, CA), ameloblastin (C17, Santa Cruz Biotechnology, Santa Cruz, CA), enamelin, phosphohistone H3 (Abcam, Cambridge, UK), keratin 14 (Abcam, Cambridge, UK) and GRP-78 (Abcam, Cambridge, UK).
Primary antibodies were detected using biotinylated secondary antibodies (Vector laboratories) followed by Cy-3-conjugated streptavidin (Sigma, Poole, UK) or an AlexaFluor488-conjugated secondary antibody (Abcam, Cambridge, UK) and the sections were mounted in fluorescence mountant containing DAPI (Vector laboratories).

We collected 10 μm-thick cryosections in accordance with standard procedure for Immunohistochemistry and EdU staining. When combined with EdU detection, we carried out firstly EdU staining using Click-iT EdU kit (Invitrogen, C10340) in accordance with the manufacturer’s instructions, then followed by a standard immunohistochemistry protocol as previously described (Fei et al., 2017 (link)). In brief, after PBS wash and permeabilization with PBST, slides were blocked in 5% serum prepared in PBST, then incubated with primary antibodies overnight at 4°C, followed by sequential PBST washes and secondary antibody (with DAPI, Sigma-Aldrich, D9542) incubation, and finally mounted using Mowiol 4-88 (Sigma-Aldrich, 9002-89-5) mounting medium after several PBST washes. Fluorescence images were acquired with an Olympus IX83 microscope (Olympus, Tokyo, Japan), using ×20 objectives and analyzed using FIJI. The following antibodies were used in this study, MBP (Genetex, GTX761141), PRRX1 (Gerber et al., 2018 (link)), βIII-TUBULIN (R&D, MAB1195), SOX9 (Chemicon, Ab5535), PAX7 (DSHB, AB528428), MEF2C (Santa Cruz, sc-365862), phospho-Histone H3 (Abcam, 10543), MHC (DSHB, A4.1025). Alexa 488-donkey-anti-mouse IgG (Jackson, 711-547-003), Alexa 555-donkey-anti-rat IgG (Invitrogen, SA5-10027), Alexa 647-donkey-anti-mouse IgG (Jackson, 715-607-003), CY3-donkey-anti-mouse IgG (Jackson, 715-165-151).

The paraffin-embedded sections of samples were baked, deparaffinized in xylene, and sequentially rehydrated in gradient concentrations of ethanol, sequentially. Then, antigen retrieval was performed in Tris-EDTA buffer (pH 9.0) heated to 95°C for 20 min. Endogenous peroxidase activity was reduced in 3% hydrogen peroxide for 10 min at room temperature. Then, 5% goat plasma was used for blocking at 37°C for 30 min, and the sections were incubated with specific primary antibodies overnight at 4°C. Primary antibodies used were Ki67 (Abcam, UK, ab279653), phospho-histone H3 (Abcam, ab267372), cleaved caspase 3 (CST, USA, #9661), GLI1 (CST, #3538), and β-catenin (CST, #8480). The following steps used biotin-streptavidin HRP detection kits (ZSGB-BIO, China, SP-9001, SP-9002) according to the manufacturer’s instructions. The sections were stained with DAB (ZSGB-BIO, ZLI-9017), counterstained with hematoxylin, dehydrated in gradient concentrations of ethanol, cleared in xylene, and mounted with neutral balsam, sequentially. Images were obtained using a slide scanner (Leica SCN400).