Eighteen somite stage embryos were fixed in 4% paraformalde-hyde overnight at 4°C, rehydrated in PBST (phosphate buffered saline + 1% Triton), incubated in immuno block (PBST + 10% normal goat serum) for 1 hr at room temperature, primaryþ antibody in immuno block overnight at 4°C, secondary antibody for 2 hr at room temperature and stored in PBST until imaged. Antibodies used: phospho-Histone H3, pHH3 (1:1,000, Cell Signaling, Cat# 3377, Danvers, MA), anti-rabbit Alexa Flour 555 (1:1,000, Cell Signaling, Cat# 4413S).
Phospho histone h3 phh3
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-Histone H3 (pHH3) is a lab equipment product that detects the phosphorylation of histone H3, a key marker of cell division and mitosis. It is a tool used in cell biology research to study cell cycle and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Poly l lysine (pll), by Merck Group (2 mentions)
Cyclin b1, by Cell Signaling Technology (2 mentions)
Cleaved parp, by Cell Signaling Technology (2 mentions)
Bond rx, by Leica (1 mentions)
Clone 5a1e, by Cell Signaling Technology (1 mentions)
Phospho cdc2 tyr15, by Cell Signaling Technology (1 mentions)
Phospho cdc25c ser216, by Cell Signaling Technology (1 mentions)
Phospho γh2a x ser139, by Cell Signaling Technology (1 mentions)
Parp1, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Super signal west chemiluminescent system, by Thermo Fisher Scientific (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Sqstm1 p62, by ABclonal (1 mentions)
4e bp1, by Cell Signaling Technology (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
P 4ebp1, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Alexa fluor 555 or 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Hnk 1, by Merck Group (1 mentions)
11 protocols using phospho histone h3 phh3
1
Immunostaining Embryonic Histone H3
2
Hematoxylin-Eosin Staining and Immunohistochemistry
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Hematoxylin and Eosin (H&E) staining: cells were incubated overnight on glass slides coated with Poly-L-lysine (Sigma) and fixed by 4% PFA in 0.1M MOPS in 0.5M NaCl, pH 7.5 for 10 min and washed with PBS. The slides were stained with Harris Hematoxylin for 5 minutes and 2% Eosin Y for 1 minute (Extended Data Fig 1e) .
Immunohistochemistry was based on Braden et al. 201445 . The labeling was done using rabbit anti Phospho-Histone H3 (pHH3; Cell Signaling, 9701) at a concentration of 1:500 (Extended Data Fig. 5d ).
Immunohistochemistry was based on Braden et al. 201445 . The labeling was done using rabbit anti Phospho-Histone H3 (pHH3; Cell Signaling, 9701) at a concentration of 1:500 (
3
Quantifying Tumor Cell Proliferation and Apoptosis
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Excised tumors were fixed in formalin for 24 h and embedded in paraffin. Subsequent processing was performed double-blind: the tissue blocks were randomly numbered for sectioning and immunohistochemistry and analysis was performed by observers blinded to treatment. The tumor containing paraffin-blocks were randomly numbered (11 treated, 11 untreated mice) and sectioned at 2.5 μm by a blinded histologist. Sections were stained with H&E and antibodies against Ki-67 (rabbit monoclonal antibody, Thermo Fisher Scientific, Boston, MA, USA; clone SP6; pretreatment Tris95 C 30′ dilution: 1:200), phosphohistone H3 (PHH3; rabbit monoclonal antibody, Cell Signaling, Beverly, MA, USA; clone 5A1E; pretreatment Tris95 C 30′ dilution: 1:100) and cleaved Caspase 3 (rabbit monoclonal antibody, Cell Signaling, clone 5A1E; pretreatment: Tris95 C 30′ dilution: 1:100). Immunohistochemistry was performed on an automated platform (Leica Bond RX, Leica Biosystems, Muttenz, Switzerland).
4
Cisplatin and MK-1775 Effects on Cell Signaling
5
Western Blot Antibodies and Protein Analysis
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The antibodies used in west blot were listed as follows: CDK6 (#db807, Diagbio, China); PARP (#9542), Cyclin D1 (#55506), Cyclin B1 (#12231), Cleaved-PARP (#5625), CDK2 (#18048), mTOR (#2983), p-mTOR (#5536), p-4E-BP1 (#2855), 4E-BP1 (#9644), Caspase-3 (#9668), phospho-Histone H3 (pHH3) (#53348), Histone H2A (γH2A.x) (#12349), Cleaved Caspase-3 (#9664), GAPDH (#5174) were purchased from Cell Signaling Technology (USA); SQSTM1/p62 (#A19700) and LC3I/LC3II (#A5618) were obtained from ABclonal Technology (Wuhan, China); goat anti-mouse and goat anti-rabbit IgG peroxidase conjugated secondary antibodies (#31460 and #31430, Thermo-Pierce, USA). The protein samples were added to 4–20% SurePAGE gels (15 wells) (GenScript, China) and transferred to PVDF membranes after protein separation. Incubate the membrane with the primary antibodies mentioned above. After washing with TBST (0.1% (v/v) Tween 20 in TBS), the membranes were incubated with secondary antibodies and then stained with enhanced chemiluminescence.
6
Hematoxylin-Eosin Staining and Immunohistochemistry
7
Immunofluorescence Analysis of Chicken Embryos
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The chicken embryos were harvested after incubation and fixed in 4% PFA overnight at 4 °C. The embryos were blocked with 10% Normal Goat Serum (SB Cat.
No. 0060-01) and incubated with the following primary antibodies at 4 °C overnight on a shaker: HNK-1 (1:500, Sigma, USA), PAX7 (1:100, DSHB, USA), AP2α (1:100, Cell Signaling Technology, USA), Phospho-Histone H3 (pHH3, 1:200, Cell Signaling Technology, USA), neurofilament (NF, 1:200, Invitrogen Antibodies, USA) and cleaved-Caspase3 (c-Caspase3, 1:200, Cell Signaling Technology, USA). After extensive rinsing in PBST (0.1% Tween-20), the embryos were incubated with the corresponding Alexa Fluor 555 or 488 secondary antibody (1:1000, Invitrogen, USA) at 4 °C overnight on a shaker. The embryos were later counterstained with DAPI (1:1000, Invitrogen, USA) at room temperature for 2 hours.
No. 0060-01) and incubated with the following primary antibodies at 4 °C overnight on a shaker: HNK-1 (1:500, Sigma, USA), PAX7 (1:100, DSHB, USA), AP2α (1:100, Cell Signaling Technology, USA), Phospho-Histone H3 (pHH3, 1:200, Cell Signaling Technology, USA), neurofilament (NF, 1:200, Invitrogen Antibodies, USA) and cleaved-Caspase3 (c-Caspase3, 1:200, Cell Signaling Technology, USA). After extensive rinsing in PBST (0.1% Tween-20), the embryos were incubated with the corresponding Alexa Fluor 555 or 488 secondary antibody (1:1000, Invitrogen, USA) at 4 °C overnight on a shaker. The embryos were later counterstained with DAPI (1:1000, Invitrogen, USA) at room temperature for 2 hours.
8
DNA Damage Response Protein Analysis
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Antibodies to p-Chk1 (#2341), 53BP1 (#4937), β-actin (#4967L), and phospho-Histone H3 (p-HH3, #9706) were purchased from Cell Signaling Technology. Antibodies to cdc25a (sc-7389) and rad51 (sc-53428) were purchased from Santa Cruz Biotechnology, and γ-H2AX (Ser139) clone JBW301 (05–636) antibody was purchased from Millipore.
9
Quantifying Cellular Proliferation and Apoptosis
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Antibody markers were analysed on seven micron, paraffin-embedded sections via standard immunofluorescence techniques using the following antibodies: Phospho-Histone H3 (pHH3) (Cell Signalling #9701, 1:100), marker of proliferation (Ki67) (BD Pharmingen #556003, 2.5 μg/ml) and cleaved caspase-3 (CC3) (abcam #ab2302, 10 μg/ml). High-temperature unmasking was performed with preheated pH 6.0 citrate buffer (Vector Labs) for 5 min, and mouse-on-mouse blocking reagent was used to block non-specific binding of mouse primary antibodies (Vector Labs). For the quantitation of positively staining pHH3, CC3 and/or Ki67 cells: tumour cell nuclei as identified by DAPI were scored for the presence or absence of pHH3, CC3 and Ki67 staining in five fields of view per tumour in triplicate for each treatment group [26 ]. GraphPad Prism 7.0a software was used to analyse immunofluorescence difference between treatment groups using a Welch’s t test for pHH3 and a Holm-Sidak corrected multiple t test for the CC3 and Ki67 co-stain.
10
Immunohistochemical and FISH Analysis of PDX Tumors
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EnaV and isotype control ADC (IgG1-b12-vcMMAE) were provided by Genmab BV (Utrecht, The Netherlands) as a stock solution (10 and 9.80 mg/mL, respectively) and diluted to a working solution of 0.8 mg/mL in phosphate buffered saline (PBS) immediately before injection. The following antibodies were used for manual immunohistochemistry (IHC) of experimental PDX tumors: alpha-smooth muscle actin (alpha-SMA; Agilent Technologies, Santa Clara, CA, USA, cat. no #M085129-2), AXL (Cell Signaling Technology, cat. no #8661), cleaved poly (ADP-ribose) polymerase (cleaved PARP; Abcam, Cambridge, UK, cat. no #32064), murine double minute 2 homolog (MDM2; ThermoFisher Scientific, Waltham, MA, USA, cat. no #337100) and phospho-histone H3 (pHH3; Cell Signaling Technology, cat. no #9701). All sections were incubated using Envision-HRP-anti-rabbit/mouse (Agilent Technologies, cat. no #K4003/#K4001), except for cleaved PARP for which SignalStain Boost IHC Detection Reagent (Cell Signaling Technology, cat. no #8125S) was used. Subsequently, stainings were developed using DAB (Agilent Technologies, cat. no #K346811), followed by hematoxylin counterstaining (VWR, Radnor, PA, USA). The following probes were used for MDM2 FISH: MDM2 (12q15)/SE12 (Leica Biosystems, Deer Park, IL, USA). Tissue pretreatment, hybridization, and detection were carried out according to the manufacturer’s instructions.
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