Phosphohistone h 3 ph 3

Phospho-histone-H3-488 (Cell Signaling), Phospho-histone-H3 (p-H3, Millipore, Billerica, MA, USA) α-tubulin (Serotec, Raleigh, NC, USA), AurKB (BD Transduction, San Jose, CA, USA), Anti-Centromere-Antibodies (ACA, Cortex Biochem, Madison, WI, USA), cyclin A and B (Santa Cruz Biotechnology, Dallas, TX, USA), and SECURIN (Gene Tex, Irvine, CA, USA). BubR1 and Bub1 were from Hongtao Yu. FITC-, Cy3-, and Cy5-conjugated secondary antibodies were from Jackson Immuno Research.

Kidneys were prepared from E16.5 embryos, newborn (NB) and adult mice (4–5 months old), fixed in 4 % paraformaldehyde and processed for tissue sections as described in [13 (link)]. Immunohistochemistry with the anti-Wnt11 antibody (Abcam) was performed using the tyramid signal amplification (TSA) kit (Perkin Elmer) as described in [13 (link)]. The Apoptosis TUNEL assays (Promega) were performed according to the manufacturer’s instruction as reported earlier [14 (link)]. Aquaporin-1 (AQP-1, Millipore), Aquaporin-2 (AQP-2, Sigma-Aldrich), thiazide-sensitive NaCl co-transporter (NCC, Millipore), acetylated α-tubulin (AT, Sigma-Aldrich), Phospho-Histone H3 (P-H3, Millipore) primary antibodies and Lotus Tetragonolobus Lectin (LTL, fluorescein labeled, Vector Laboratories), Dolichos Biflorus Agglutinin (DBA, rhodamine labeled, Vector Laboratories) lectins were used according to the manufacturers’ recommendations. Alexa Fluor 488 and 546-conjugated antibodies (Invitrogen) served as the secondary antibodies. DAPI (Sigma Pharmaceuticals) was used to stain the nuclei of the cells in the tissue sections. Electron microscopy samples were prepared as previously described [15 (link)] and examined using Phillips CM100 transmission electron microscope.

Embryonic brain sections were washed in PBS, blocked in a solution of 5% normal goat serum (Merck) (v/v) containing 0.1% Triton X-100 (v/v) (Merck) in PBS at RT for 2 h. They were subsequently incubated in primary antibodies at RT for 2 h and then at 4 °C overnight. The following antibodies were used: mouse monoclonal Nestin (1:100, DSHB) and 5-Bromodeoxyuridine (BrdU; 1:1000, Progen), Cad8-1 (1:100, DSHB), chicken polyclonal raised against GFP (1:500, Aves Laboratories), rabbit polyclonal raised against calbindin (CB-28; 1:3000, Swant), cleaved caspase-3 (CC3; 1:250, Cell Signaling Technology), Ki67 (1:1000, Cell Signaling), Tbr2 (1:500, Abcam) or phosphohistone H-3 (PH-3; 1:1000, Millipore). For blood vessel staining, sections were incubated with biotinylated Griffonia (Bandeiraea) Simplicifolia lectin I (Isolectin B4) (1:200, Vector) followed by fluorescent Strepatividin-405 (1:200, Vector Laboratories).

The hearts were optimal cutting temperature compound and cut into 4 μm sections. CMs and tissues sections while frozen were fixed with 4% paraformaldehyde (Solarbio, China). Then, the cells were permeabilized with 0.4% Triton X-100 (Solarbio, China) in PBS, blocked with goat serum (Boster, China) and incubated with primary antibodies against phospho-Histone H3 (pH3; Millipore, USA), Ki67 (Abcam,UK), α-actinin (Abcam, UK), vimentin (Abcam, UK) at 4 °C overnight followed by Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibody for 1h. The cells were counterstained with DAPI (Solarbio, China) to label the nuclei.

Cells were fixed with 4% fixative solution (P1110, Solar IR) for 15 min at room temperature and then penetrated with 0.4% Triton-X100 (dissolved in 5 mg/mL BSA) for 90 min at room temperature. The cells were then blocked with goat serum (AR0009, Boster) for 30 min at 37 °C and incubated with antibodies to α-actinin (GTX29465, Gene Tex, 1:400), phosphoHi-stone H3 (pH3; #06–570, Millipore, Billerica, MA, USA, 1:400), Ki67(ab15580, Abcam, Cambridge, UK, 1:400) overnight at 4 °C. The secondary antibodies Alexa Fluor 488 (ab150113, Abcam, 1:400) and Alexa Fluor 594 (ab150080, Abcam, 1:400) were incubated with cells for 60 min at room temperature in the dark. After mounting and nucleic staining with DAPI (C0065; Solarbio, Beijing, China) for 15 min at room temperature, images were captured using confocal laser scanning microscope (FV300; Olympus, Japan) and analyzed with ImageJ software.
To detect cell proliferation, cells were incubated with 5-ethyl-2 ‘-deoxyuridine (EdU) for 12 h and then fixed, penetrated, and blocked as described above. Cardiomyocytes were counter-stained with α-actinin (GTX29465, Gene Tex, 1:400). The dye solution was prepared with EdU Apollo567 in Vitro Kit (Ribobio, Guangzhou, China) according to the instructions, and the cells were incubated in the dark for 30 min at room temperature. The nucleus is labeled with DAPI.