Ab5176

Manufactured by Abcam

Sourced in United States

Ab5176 is a laboratory equipment product. It is a core function component for research and scientific applications.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions) Ab16667, by Abcam (3 mentions) Ab150077, by Abcam (3 mentions) Ab15580, by Abcam (2 mentions) Ab70369, by Abcam (2 mentions) Alexa fluor 647, by Thermo Fisher Scientific (2 mentions) T9028, by Merck Group (2 mentions) T6793, by Merck Group (2 mentions) Alexafluor 488 conjugated phallacidin, by Thermo Fisher Scientific (2 mentions) A7811, by Merck Group (2 mentions) Ab32053, by Abcam (2 mentions) M6a seq, by Synaptic Systems (2 mentions) M6a clip seq, by Synaptic Systems (2 mentions) A5892, by Merck Group (2 mentions) Ab11174, by Abcam (2 mentions) Ab2175, by Abcam (2 mentions) Ab26350, by Abcam (2 mentions) Ab1791, by Abcam (2 mentions) Ab189849, by Abcam (2 mentions) Ab9110, by Abcam (2 mentions)

42 protocols using ab5176

Immunofluorescence and Immunohistochemistry for Proliferation

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Zebrafish embryos were collected at 48 hpf to perform whole‐mount immunofluorescence. After fixing in 4% paraformaldehyde (PFA) at 4°C overnight, digestion using Collagenase, Type II (Life technologies), and blocking at room temperature, the embryos were incubated with the rabbit anti‐Histone H3 (phospho S10) (ab5176, Abcam) and mouse anti‐BrdU (sc‐32323, Santa Cruz Biotechnology). For BrdU assay, embryos were exposed to BrdU for 6 h before collection.
For tissue sections of mouse hearts, the proliferation of cardiomyocytes was analysed by immunohistochemical staining using rabbit anti‐Ki67 (ab15580, Abcam) and rabbit anti‐Histone H3 (phospho S10) (ab5176, Abcam). To identify the positivity of the immune‐stained sections, negative controls (NCs) were set that the slices from the same batch were only incubated with isotype matched secondary antibodies without binding with specific primary antibodies.

Hao L., Ma J., Wu F., Ma X., Qian M., Sheng W., Yan T., Tang N., Jiang X., Zhang B., Xiao D., Qian Y., Zhang J., Jiang N., Zhou W., Chen W., Ma D, & Huang G. (2022). WDR62 variants contribute to congenital heart disease by inhibiting cardiomyocyte proliferation. Clinical and Translational Medicine, 12(7), e941.

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Quantifying SKIV2L2 and H3 phospho-S10 by Western Blot

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Protein extracts in Laemmli buffer were run at 100V on a 10% acrylamide (37.5:1) SDS-PAGE gel. Acid-extracted nuclear fractions and cytoplasmic fractions were run on separate gels on the same day. Proteins were then transferred onto BioTrace nitrocellulose membrane, 0.2 µm pore size (Pall Corporation #66485) at 4°C and 15 V for 12 h. The membrane was incubated in blotting milk containing polyclonal antibodies raised in rabbit against mouse SKIV2L2 (1:1000 dilution, Abcam ab187884), β-actin (1:50 dilution, Thermo Fisher #PA5-16914), or H3 phospho-S10 (1:500 dilution, Abcam ab5176) for 16 h at 4°C, followed by goat anti-rabbit antibody conjugated to horseradish peroxidase (1:5,000 dilution, Abcam ab6721) Protein was detected using luminol, ρ-coumaric acid, and hydrogen peroxide and capturing chemiluminescence with the UVP Biospectrum System. SKIV2L2 and H3 phospho-S10 protein levels were quantified by normalizing to ACTB using the ImageJ software provided by the NIH. A Student's t-test measured significance between control and Skiv2l2 siRNA samples, while a one-way ANOVA with Tukey's HSD test was used with comparison to chemically induced differentiation.

Onderak A.M, & Anderson J.T. (2017). Loss of the RNA helicase SKIV2L2 impairs mitotic progression and replication-dependent histone mRNA turnover in murine cell lines. RNA, 23(6), 910-926.

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Immunofluorescence Staining of DNA Damage Markers

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Cells were grown on sterile 12-mm glass coverslips, fixed in 3% formaldehyde in PBS for 15 min at room temperature, washed once in PBS, permeabilized for 5 min at room temperature in PBS supplemented with 0.2% Triton X-100 (Sigma-Aldrich), and washed twice in PBS. All primary antibodies (see below for specifications) and secondary antibodies (Alexa fluorophores; Life Technologies) were diluted in filtered DMEM containing 10% FBS and 0.02% sodium azide. Antibody incubations were performed for 1–2 h at room temperature. After antibody incubations, coverslips were washed once with PBS and incubated for 10 min with PBS containing DAPI (0.5 µg/ml) at room temperature to stain DNA. Following three washing steps in PBS, coverslips were briefly washed with distilled water and mounted on 6 µl Mowiol-based mounting media. The following primary antibodies were used for immunostaining: CHK1 (1:500; rabbit; ab40866; Abcam), CHK1 phospho S296 (1:500; rabbit; ab79758; Abcam), H2AX phospho S139 (1:1,000; mouse; 613401; Biolegend), KAP1 phospho S824 (1:500; rabbit; ab70369; Abcam), RPA32 phospho S4/8 (1:500; rabbit; A300-245A; Bethyl), p21 (1:100; rabbit; sc-756; Santa Cruz), HA (1:500; mouse; 901513; Biolegend), and pH3S10 (rabbit; ab5176; Abcam).

Michelena J., Gatti M., Teloni F., Imhof R, & Altmeyer M. (2019). Basal CHK1 activity safeguards its stability to maintain intrinsic S-phase checkpoint functions. The Journal of Cell Biology, 218(9), 2865-2875.

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Immunohistochemistry for Larval Development

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Postlarvae and juveniles on the coverslips were fixed according to [46 ]. Immunohistochemistry followed the protocol described in [28 (link)], using the antibodies against phospho-histone H3 [pSer10] (rabbit, 1:500, Abcam ab5176), acetylated-∂-tubulin (mouse 1:500, Sigma-Aldrich T6793) and tyrosinated-∂-tubulin (mouse 1:500, Sigma-Aldrich T9028). For secondary antibodies, we used AlexaFluor 488 (anti-rabbit or anti-mouse. 1:200, Molecular Probes), AlexaFluor 568 (anti-rabbit or anti-mouse. 1:200, Molecular Probes) and AlexaFluor 647 (anti-rabbit or anti-mouse, 1:200, Molecular Probes). AlexaFluor 488-conjugated phallacidin (1:25, Molecular Probes), which is generally used to label filamentous actin, was used as a counterstain to label F-actin-enriched cells in the inner cell mass and epithelial layer in larvae. For all samples, nuclei were labeled with the fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI; 1:1000, Molecular Probes) for 30 min, washed in PBST for 5 min and mounted using ProLong Gold anti-fade reagent (Molecular Probes). All samples were observed using the Zeiss LSM 510 META confocal microscope, and image analysis was performed using the software ImageJ.

Sogabe S., Nakanishi N, & Degnan B.M. (2016). The ontogeny of choanocyte chambers during metamorphosis in the demosponge Amphimedon queenslandica. EvoDevo, 7, 6.

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Inhibiting miR-31a-5p in Neonatal Rat Hearts

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miR-31a-5p antagomir (2‘OME+ 5’chol modified) (RiboBio) injections were performed. To inhibit miR-31a-5p in neonatal rats, 50 mg kg⁻¹ antagomir or the scramble control was given intraperitoneally for 3 consecutive days after birth, and 5-ethynyl-2′-deoxyuridine (EdU, 50 mg kg⁻¹) was injected intraperitoneally daily 2 days before harvesting at P4. Ventricular tissues were snap frozen in liquid nitrogen with OCT on the short axis at 8 μm. For staining, sections were fixed in 4% paraformaldehyde (PFA) followed by washing with PBS. Sections were blocked with 3% (w/v) BSA in PBS and then incubated with primary antibodies overnight: p-histone H3 (1:100, ab5176, Abcam), Ki-67 (1:100, ab 16667, Abcam) and α-actinin (1:100, A7811, Sigma-Aldrich).

Xiao J., Liu H., Cretoiu D., Toader D.O., Suciu N., Shi J., Shen S., Bei Y., Sluijter J.P., Das S., Kong X, & Li X. (2017). miR-31a-5p promotes postnatal cardiomyocyte proliferation by targeting RhoBTB1. Experimental & Molecular Medicine, 49(10), e386-.

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Immunofluorescence Analysis of Cell Cycle Markers

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Cells from each group were fixed in 4% (vol/vol) paraformaldehyde for 30 minutes. Cells were incubated overnight at 4°C with oscillation with primary antibody against either histone H₃ (phospho Ser10) (1:300, ab5176; Abcam), Cdc2 (1:300, ab18; Abcam), Cdc2 (phospho Tyr15) (1:200, ab18; Abcam) or cyclin B1 (1:100, ab32053; Abcam). Then, cells were incubated in the dark for 1 hour at room temperature using Alexa Fluor 488 conjugated secondary antibody (1:200; Invitrogen, USA). Cells were counterstained for 10 minutes in the dark with the nuclear dye Hoechst (Hoechst AG, Germany) diluted 1:4000 in PBS. The fluorescence was captured and observed using an inverted microscope linked to a laser scanning confocal microscope (FluoView1000; Olympus).18, 38

Sun Z., Li Y., Wang H., Cai M., Gao S., Liu J., Tong L., Hu Z., Wang Y., Wang K., Zhang L., Cao X., Zhang S., Shi F, & Zhao J. (2019). miR‐181c‐5p mediates simulated microgravity‐induced impaired osteoblast proliferation by promoting cell cycle arrested in the G2 phase. Journal of Cellular and Molecular Medicine, 23(5), 3302-3316.

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Protein Expression Analysis in Kidney Samples

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Protein samples were extracted from the kidneys. The lysates (50 µg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis with a 10% (wt/vol) acrylamide gel and transferred to polyvinylidene fluoride membranes. After blocking with 10% milk and incubating with primary and secondary antibodies, the blots were visualized by enhanced chemiluminescence. The intensity of each band was quantified by densitometry with Image Lab 5.2.1 and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) before relative quantification. The primary antibodies used in our study included Nrf2 (ab62352, Abcam, Cambridge, UK), heme oxygenase 1 (HO-1; ab13248, Abcam), GAPDH (60004-1-Ig, Proteintech, Wuhan, China), Histone 3 (ab5176, Abcam), phosphorylated (p)-I-kappa-B-alpha (IκBα) (#2859, Cell Signaling Technology, Danvers, MA), IκBα (#4812, Cell Signaling Technology), p-P65 (BS4135, Bioworld, Dublin, OH), p65 (BS3648, Bioworld), transforming growth factor (TGF)-β1 (ab92486, Abcam), p-Smad1/5 (#9516, Cell Signaling Technology), p-Smad2 (#18338, Cell Signaling Technology), IL-6 (#12912, Cell Signaling Technology), and TNF-α (ab34674, Abcam).

Yang Z.J., Wang H.R., Wang Y.I., Zhai Z.H., Wang L.W., Li L., Zhang C, & Tang L. (2019). Myricetin Attenuated Diabetes-Associated Kidney Injuries and Dysfunction via Regulating Nuclear Factor (Erythroid Derived 2)-Like 2 and Nuclear Factor-κB Signaling. Frontiers in Pharmacology, 10, 647.

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Antibodies for Cell Signaling Analysis

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Antibodies against Cnot1, Cnot2, Cnot6L, Cnot7, Cnot8 and Cnot9 have been described previously (Suzuki et al., 2015 (link)). Antibodies against α-tubulin (DM1A, sc-32293), Cdt1 (sc-365305), Brca1 (sc-135731), Alb (sc-46291, for WB), normal mouse IgG (sc-2025) and normal rabbit IgG (sc-2026) were purchased from Santa Cruz Biotechnology. Antibodies against cleaved caspase-3 (9664) and p53 (2524) were from Cell Signaling Technology. Anti-Ki67 rabbit monoclonal antibody for immunohistochemistry was from NeoMarkers (RM-9106-S0). Antibodies against CK19 (ab133496), Ki67 (ab16667), phospho-histone H3 (Ser10) (ab5176), Afp (ab46799), Igf2 (ab9574), CD45 (ab10558) and F4/80 (ab111101) were from Abcam. Anti-Alb antibody (MAB-1455, for immunofluorescence) was from B&D systems. Antibodies against Chk1 (K0086-3) and Iqgap1 (K0100-3) were from MBL. Anti-Klf6 antibody (14716-1-AP) was from Proteintech.

Suzuki T., Kikuguchi C., Nishijima S., Nagashima T., Takahashi A., Okada M, & Yamamoto T. (2019). Postnatal liver functional maturation requires Cnot complex-mediated decay of mRNAs encoding cell cycle and immature liver genes. Development (Cambridge, England), 146(4), dev168146.

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Srek1L Regulates Hepatocyte Proliferation

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Liver regeneration after partial hepatectomy (PHx) may facilitate tumor growth and HCC recurrence. It remodels a microenvironment through alterations in cellular signaling pathways, which activate the proliferation of mature hepatocytes^{60 (link)}. Thus, we utilized PHx to evaluate the effect of Srek1^L on hepatocyte proliferation. Gain-of-function experiments were performed through overexpressing Srek1^L in C57BL/6 mice by intravenous tail vein injection with control (AAV8-CMV-eGFP) or with Srek1^L overexpressing adeno-associated virus (AAV8-CMV-SREK1-IRES-eGFP) (Hanbio Biotechnology, assay ID: HH20210414HZCY-AAV01) with a titer 1 × 10¹² vg ml⁻¹. To evaluate the mRNA level of Srek1^L, mice were sacrificed 40 days post-AAV injection, and livers were isolated for GFP fluorescence detection and RNA extraction. GFP fluorescence detection was carried out by PhotoIMAGER OPTIMA (Biospace lab, Paris, France).). Srek1^L mRNA level is detected by real-time PCR. Mice livers were collected, frozen and embedded in OCT (Sakura 4583) compound 4th and 8th days post PHx. Livers blocks were sectioned and stained by phosphorylation of histone 3 serine 10 (anti-pH3S10; Abcam ab5176; 1:200) for proliferative potential evaluation.

Chang C., Rajasekaran M., Qiao Y., Dong H., Wang Y., Xia H., Deivasigamani A., Wu M., Sekar K., Gao H., Sun M., Niu Y., Li Q., Tao L., Yan Z., Wang M., Chen S., Zhao S., Chen D., Li L., Yang F., Gao H., Chen B., Su L., Xu L., Chen Y., Seshachalam V.P., Chen G., Gunaratne J., Hong W., Shi J., Chen G., Grierson D.S., Chabot B., Xie T., Hui K.M, & Chen J. (2022). The aberrant upregulation of exon 10-inclusive SREK1 through SRSF10 acts as an oncogenic driver in human hepatocellular carcinoma. Nature Communications, 13, 1363.

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Histological and Immunofluorescent Analysis of Cultured Hindlimbs

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Cultured hindlimbs were processed in a sucrose gradient (15% and then 30%) and embedded in a 30% sucrose and optimal cutting temperature compound 50:50 mix. Ten-micrometer sections of the medial condyles with full-length femora and lateral condyles were collected along the sagittal axis of the femora and fixed in 4% paraformaldehyde.
For histology, frozen sections were stained with Toluidine blue (sGAG stain) for 4 min and then washed with deionized water or stained in Picrosirius red (collagen stain) for 30 min, followed by 10 min in acidified water. Sections were imaged using light microscopy with a consistent exposure time (Yenway EX30l; Life Sciences Microscope, Glasgow, UK). For immunofluorescence, frozen sections were washed with PBTD (0.1% Tween 20 and 1% DMSO in phosphate-buffered saline) for permeabilization, blocked with 5% normal goat serum for 2 hours, incubated with primary antibodies against TRPV4 protein (1:500; ab39260, Abcam) or pHH3 (1:500; ab5176, Abcam) at 4°C overnight, followed by Alexa Fluor 488–conjugated secondary antibody (1:200; ab150077, Abcam) for 2 hours and lastly stained with 4′,6-diamidino-2-phenylindole (DAPI) for 3 min.

Khatib N.S., Monsen J., Ahmed S., Huang Y., Hoey D.A, & Nowlan N.C. (2023). Mechanoregulatory role of TRPV4 in prenatal skeletal development. Science Advances, 9(4), eade2155.

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