rabbit primary antibody against phospho histon 3 ph3  (Cell Signaling Technology Inc)

Journal: Cell Death & Disease

Article Title: CUGBP1, a crucial factor for heart regeneration in mice

doi: 10.1038/s41419-022-04570-w

Figure Lengend Snippet: A CUGBP1 was overexpressed in the 14-day old mice hearts and apical resection was performed simultaneously. Seven days after resection, cardiac tissue from apex zone was collected for paraffin section immunofluorescence detection of PH3 (red) and TNNI (green). Representative images are presented. B Statistical analysis of the number of PH3/TNNI-positive cells in apex and remote zone in the 14-day old mice heart following the Ad-virus infection and resection on seven days after resection. C Statistical analysis of the number of PH3/TNNI-positive cells by paraffin sections for immunofluorescence in the 14-day old mice heart following the Ad-virus infection and resection. Scale bars: 50 μm, 10 μm(inset). Data are presented as the mean ± SEM ( n = 8, 9 fields each mouse). * p < 0.05, ** p < 0.01 vs. control. dpi&r, days post injection & resection.

Article Snippet: Following deparaffinization and high-pressure antigen retrieval with 10 mM sodium citrate buffer (PH 6.0) in boiling water for 2 min, paraffin sections were blocked with 10% goat serum (ZLI-9022, ZSGB-BIO, China) for 1 h. Primary antibodies against PH3 (ab177218, abcam), ki67 (ab16667, abcam), CD31 (ab28364, abcam) and cTnI (66376-1-Ig, proteintech) were incubated overnight at 4 °C.

Techniques: Paraffin Section, Immunofluorescence, Virus, Infection, Control, Injection

Journal: Development (Cambridge, England)

Article Title: Myc activity is required for maintenance of the neuromesodermal progenitor signalling network and for segmentation clock gene oscillations in mouse

doi: 10.1242/dev.161091

Figure Lengend Snippet: cMyc is co-expressed with Wnt3a , Fgf8 , Sox2 and brachyury in the CLE. (A) Representative confocal images of an E8.5 embryo labelled by immunofluorescence for Sox2 and brachyury ( n =3 embryos). (a) Whole-mount E8.5 embryo and (b-d) transverse sections at the level of the CLE domain (demarcated by the white dashed line in a). Sox2 labels the neuroepithelium along the anterior-posterior axis and the CLE, whereas brachyury labels the PSM and tail bud mesoderm. (e) Magnification of d, showing the location of the NMPs (Sox2/brachyury co-expressing cells) in the CLE epithelium. (B) Representative in situ hybridization (ISH) images of E8.5 embryos labelled for (a) cMyc ( n =10 embryos), (b) MycN ( n =4 embryos), (c) Fgf8 ( n =3 embryos), (d) Wnt3a ( n =3 embryos) and (e) Lfringe ( n =5 embryos). (a′-e′) Transverse sections of the CLE and underlying cPSM domain indicated by the white dashed lines in a-e. (a′) cMyc , (c′) Fgf8 and (d′) Wnt3a show high levels of expression in the CLE. (b′) MycN and (e′) Lfringe show high levels of expression in the cPSM. (C) Representative confocal images of immunofluorescence labelling of E8.5 embryos for cMyc and Sox2 (a-g; n =3 embryos) and cMyc and brachyury (h-n; n =3 embryos). Sox2/cMyc co-expressing cells are evident in the transverse sections of the CLE (e-g; panels correspond to sections at the level of the domain demarcated by the white dashed line in a). Brachyury/cMyc co-expressing cells are evident both in the CLE and underlying cPSM (l-n; panels correspond to the white dashed line in e). Scale bars: 100 μm.

Article Snippet: E8.5 whole mount, cryosections or explants of embryos were stained for immunofluorescence using primary antibodies against Sox2 [neural marker, raised in goat (Immune Systems, GT15098; LOT 909901)], brachyury [mesoderm marker, raised in rabbit (Santa Cruz Biotechnology, H-210; LOT H2514); or raised in goat (R&D Systems, AF2085; LOT KQP0319031)] and pH3 (Upstate Cell Signaling Solutions, 06-570; LOT32219). cMyc-positive cells were labelled using the monoclonal anti-c-Myc antibody [raised in rabbit (Abcam, Y69; LOT GR255057-5)].

Techniques: Immunofluorescence, Expressing, In Situ Hybridization

Journal: Development (Cambridge, England)

Article Title: Myc activity is required for maintenance of the neuromesodermal progenitor signalling network and for segmentation clock gene oscillations in mouse

doi: 10.1242/dev.161091

Figure Lengend Snippet: cMyc expression persists in the tail bud during E9.5-E10.5 and shows dynamic expression at the transcript level. (A) Representative ISH images of a tail bad at E9.5 and E10.5. (a) Whole-mount E9.5 embryo labelled for cMyc at E9.5 ( n =12 embryos). (b,b′) High levels of cMyc are present in the caudal-most neuroepithelium and adjacent PSM (arrowheads). (c) Side view of an E10.5 tail labelled for cMyc mRNA. (d) Three different expression profiles for cMyc in the PSM of E10.5 embryos, reminiscent of the three phases of segmentation clock gene expression ( n =10 embryos). (B) Representative confocal images of immunofluorescence labelling for cMyc in E9.5 and E10.5 embryos. (a-f) cMyc and brachyury staining in whole-mount embryos. (a′-f′) Higher magnification images of a-f showing cMyc/brachyury co-expressing cells in the tail bud ( n =5 embryos). (g-l) cMyc and Sox2 labelling in whole-mount embryos. (g′-l′) Higher magnification images of g-l, showing cMyc/Sox2 co-expressing cells in the tail bud ( n =5 embryos). Scale bars: 100 μm.

Article Snippet: E8.5 whole mount, cryosections or explants of embryos were stained for immunofluorescence using primary antibodies against Sox2 [neural marker, raised in goat (Immune Systems, GT15098; LOT 909901)], brachyury [mesoderm marker, raised in rabbit (Santa Cruz Biotechnology, H-210; LOT H2514); or raised in goat (R&D Systems, AF2085; LOT KQP0319031)] and pH3 (Upstate Cell Signaling Solutions, 06-570; LOT32219). cMyc-positive cells were labelled using the monoclonal anti-c-Myc antibody [raised in rabbit (Abcam, Y69; LOT GR255057-5)].

Techniques: Expressing, Gene Expression, Immunofluorescence, Staining

Journal: Development (Cambridge, England)

Article Title: Myc activity is required for maintenance of the neuromesodermal progenitor signalling network and for segmentation clock gene oscillations in mouse

doi: 10.1242/dev.161091

Figure Lengend Snippet: Myc activity suppression results in downregulation of Wnt3a/8a , Fgf8 and Sox2 . (A) Representative ISH images of CLE/cPSM explants treated with DMSO (a-f) or 10 μM JQ1 (a′-f′) for 6 h. Fgf8 (a,a′; n =8/8 embryos), Wnt3a (b,b′; 5/5 embryos) and Sox2 (f,f′; 3/3 embryos) expression is suppressed upon JQ1 treatment. In contrast, expression of Axin2 (c,c′; n =4/4 embryos), Sprouty2 (d,d′; n =4/4 embryos) and brachyury (e,e′; n =6/6 embryos) is not affected. (B) Representative ISH images of half-tail explants from E8.5 embryos (micro-dissected below the level of the last somite pair), treated either with DMSO or 10 μΜ JQ1 for 6 h, showing no effect on expression of the Notch target gene Lfringe ( n =4/4 embryos). RT-qPCR analysis of CLE/cPSM explants for Hes5 expression show no differences upon treatment with 10 μΜ JQ1 or 75 μΜ 10074G5 for 6 h. (C) Characterization of gene expression changes in CLE/cPSM explants upon 10 μΜ JQ1 or 75 μΜ 10074G5 for 6 h. Relative gene expression, normalized to actin levels, is shown. Data are from three independent experiments, presented as mean±s.e.m. Statistical significance was assessed using the unpaired two-tailed Student’s t -test for samples with unequal variance. Scale bars: 100 μm.

Article Snippet: E8.5 whole mount, cryosections or explants of embryos were stained for immunofluorescence using primary antibodies against Sox2 [neural marker, raised in goat (Immune Systems, GT15098; LOT 909901)], brachyury [mesoderm marker, raised in rabbit (Santa Cruz Biotechnology, H-210; LOT H2514); or raised in goat (R&D Systems, AF2085; LOT KQP0319031)] and pH3 (Upstate Cell Signaling Solutions, 06-570; LOT32219). cMyc-positive cells were labelled using the monoclonal anti-c-Myc antibody [raised in rabbit (Abcam, Y69; LOT GR255057-5)].

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Gene Expression, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: Myc activity is required for maintenance of the neuromesodermal progenitor signalling network and for segmentation clock gene oscillations in mouse

doi: 10.1242/dev.161091

Figure Lengend Snippet: Myc activity suppression does not promote differentiation, nor does it compromise viability and proliferation. (A) Representative confocal images of CLE/cPSM explants cultured for 10 h, either in DMSO or in 10 μΜ JQ1 and subsequently stained for pH3 by immunofluorescence ( n =3 embryos). The TUNEL assay was employed to analyse cell death. Apoptotic nuclei were detected in peripheral edges of both DMSO- and JQ1-treated explants ( n =3 embryos). (B) No significant differences in the number of pH3-positive loci were found between DMSO- and JQ1-treated explants (explants micro-dissected from three different embryos, quantified in 18 optical sections per condition). Data are presented as mean±s.e.m. (C) CLE/cPSM explants treated in DMSO (a-d) or with 10 μΜ JQ1 (a′-d′) for 10 h analysed by ISH. At this timeframe, both Sox2 ( n =3/3) and brachyury ( n =7/10) expression is downregulated, and differentiation markers such as Pax6 ( n =0/5 embryos) and Paraxis ( n =0/3 embryos) are not expressed in control DMSO and JQ1-treated explants. (D) CLE/cPSM explants after 10 h of culture in DMSO (a-d) or in 10 μΜ JQ1 (a′-b′). Representative ISH images of explants treated with CT99021 (a,b,a′,b′) or 100 nM RA (c,d,c′,d′) for 14 h or 20 h following the 10 h culture in DMSO or 10 μΜ JQ1. Transient Myc suppression for 10 h prior to 14 h of CT99021 stimulation (a′) causes low levels of Tbx6 mRNA ( n =6/6 embryos) compared with DMSO treatment alone (a′). High levels of Tbx6 when CT9901 exposure was prolonged to 20 h ( n =4/4 embryos) (b-b′). Explants transiently incubated with JQ1 express no Pax6 after 14 h of RA treatment ( n = 0/6 embryos), in contrast to the DMSO control explants. Explants transiently incubated with JQ1 do express Pax6 after 20 h of RA treatment ( n = 5/5 embryos), as do the control explants. (E) RT-qPCR analysis of gene expression changes in control CLE/cPSM explants cultured in DMSO or in 10 μΜ JQ1 for 10 h. Relative gene expression, normalized to actin levels, is shown. Data are from three independent experiments, presented as mean±s.e.m. Statistical significance was assessed using the unpaired two-tailed Student’s t -test for samples with unequal variance. * P <0.05, ** P <0.01, *** P <0.001; ns, nonsignificant. Scale bars: 100 μm.

Article Snippet: E8.5 whole mount, cryosections or explants of embryos were stained for immunofluorescence using primary antibodies against Sox2 [neural marker, raised in goat (Immune Systems, GT15098; LOT 909901)], brachyury [mesoderm marker, raised in rabbit (Santa Cruz Biotechnology, H-210; LOT H2514); or raised in goat (R&D Systems, AF2085; LOT KQP0319031)] and pH3 (Upstate Cell Signaling Solutions, 06-570; LOT32219). cMyc-positive cells were labelled using the monoclonal anti-c-Myc antibody [raised in rabbit (Abcam, Y69; LOT GR255057-5)].

Techniques: Activity Assay, Cell Culture, Staining, Immunofluorescence, TUNEL Assay, Expressing, Control, Incubation, Quantitative RT-PCR, Gene Expression, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: Myc activity is required for maintenance of the neuromesodermal progenitor signalling network and for segmentation clock gene oscillations in mouse

doi: 10.1242/dev.161091

Figure Lengend Snippet: Conditional inducible cMyc depletion from the CLE results in downregulation of Fgf8 expression during axis elongation. (A) (a-f) Representative images of E10.5 control embryos ( n =3 embryos) labelled by ISH for Fgf8 expression show high expression levels in the hind limbs and in the tail bud. (c) Transverse section in the limb bud region demarcated by the white dashed line in b. (d-f) Transverse sections of the CNH and tail bud demarcated by the white dashed line box in b. (g-r) Confocal images of transverse sections of the CNH and tail bud labelled by immunofluorescence for Sox2 and brachyury, showing a large number of co-expressing cells in the tail bud mesenchyme (data from ten confocal sections from two embryos). (B) Representative images of E10.5 cMyc conditional inducible mutant embryos ( n =3 embryos) labelled by ISH for Fgf8 expression. (a′-f′) High levels of Fgf8 are detected in the hind limbs, whereas very low levels are detected in the tail bud. (c′) Transverse section in the limb bud region demarcated by the white dashed line box in b′. (d′-f′) Transverse sections of the CNH and tail bud demarcated by the white dashed line box in b′. (g′-z′) Confocal images of transverse sections of the CNH and tail bud labelled by immunofluorescence for Sox2, brachyury and GFP show large numbers of co-expressing cells in the tail bud mesenchyme. GFP cells are mostly absent from the CNH (s′), where Fgf8 expression is still detected (f′) (data from ten confocal sections from two embryos). Scale bars: 100 μm. (C) Quantification of Sox2/brachyury co-expressing tail bud cells in control ( n =2; eight sections) and cMyc KO ( n =2; eight sections) embryos did not reveal differences. The counts are normalized to the total number of DAPI-stained nuclei. (D) Measurements of the neural tube (NT) area and cell density did not reveal differences between control ( n =2; 25 sections) and cMyc KO embryos ( n =2; 18 sections). (E) No differences between somite number (counted below hind limb level) or size were found between control ( n =5) and cMyc KO embryos ( n =8).

Article Snippet: E8.5 whole mount, cryosections or explants of embryos were stained for immunofluorescence using primary antibodies against Sox2 [neural marker, raised in goat (Immune Systems, GT15098; LOT 909901)], brachyury [mesoderm marker, raised in rabbit (Santa Cruz Biotechnology, H-210; LOT H2514); or raised in goat (R&D Systems, AF2085; LOT KQP0319031)] and pH3 (Upstate Cell Signaling Solutions, 06-570; LOT32219). cMyc-positive cells were labelled using the monoclonal anti-c-Myc antibody [raised in rabbit (Abcam, Y69; LOT GR255057-5)].

Techniques: Expressing, Control, Immunofluorescence, Mutagenesis, Staining