After 4-µm kidney sections were deparaffinized and hydrated, each section was exposed to 3% hydrogen peroxide solution. After antigen repair, kidney sections were incubated with primary antibodies against γ H2AX (1:200, Abcam, ab2893) overnight at 4°C. Subsequently, sections were incubated with HRP-conjugated secondary antibody (Proteintech Group, Inc., Chicago, IL, United States) at room temperature for 30 min; a DAB kit (Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) was used for localization. Images were taken through a microscope (×400).
Ab2893
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab2893 is a primary antibody product available from Abcam. It is a rabbit polyclonal antibody that recognizes a specific target. The core function of this product is to detect and bind to the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Ab8898, by Abcam (5 mentions)
Ab11175, by Abcam (5 mentions)
Ab1791, by Abcam (4 mentions)
Ab36823, by Abcam (4 mentions)
Ab1220, by Abcam (3 mentions)
Ab150077, by Abcam (3 mentions)
Ab97672, by Abcam (3 mentions)
Ab13840, by Abcam (3 mentions)
Ab80779, by Abcam (3 mentions)
Ab15093, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab4729, by Abcam (2 mentions)
Ab37438, by Abcam (2 mentions)
Ab20669, by Abcam (2 mentions)
Ab6002, by Abcam (2 mentions)
Ne per nuclear and cytoplasmic extraction reagents kit, by Thermo Fisher Scientific (2 mentions)
Coomassie protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce mitochondria isolation kit, by Thermo Fisher Scientific (2 mentions)
Ab4340, by Merck Group (2 mentions)
Ha sc 7392, by Santa Cruz Biotechnology (2 mentions)
91 protocols using ab2893
1
Quantifying DNA Damage in Kidney Tissue
2
PTEN-mediated DNA Damage Response
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HCT116 PTEN-null cells were seeded on Lab-Tek eight-well chamber coated with laminin (L2020, Sigma) and incubated overnight. Cells were treated with 1 mM hydroxyurea (H8627, Sigma) for 16 h after a 24-h transfection with either wild-type PTEN or mutated constructs. Cells were fixed as previously described16 (link) and immunolabeled with a rabbit anti-phosphorylated histone H2AX (γH2AX) antibody (ab2893, Abcam) and a anti-rabbit IgG Alexa 568 antibody (Invitrogen). Images were acquired by confocal fluorescence microscopy (Zeiss LSM 780 NLO) equipped with a 63× objective. The numbers of nuclear foci of phosphorylated histone H2AX were manually counted. Nuclei were identified by staining with Hoechst 33258 (Molecular Probes).
3
Chromatin Modifications in MEF Cells
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All cell lines were cultured in DMEM medium with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (Millipore-Sigma) in a humidified incubator with 5% CO2 at 37 °C.
Initial experiments were performed on primary MEFs derived from 13.5 days NM1 WT and KO embryos46 (link). In other experiments were used immortalized MEFs (ATCC® CRL-2752) or cell lines derived from these immortalized cells.
The antibodies against H3K9me3 (ab8898), H3K27ac (ab4729), H3K4me1 (ab8895), H3K4me3 (ab8580), H3K9ac (ab10812), H3 (ab1791), γH2AX (ab2893), p53-K370 (ab183544), PCAF (ab12188), Set1 (ab70378), GAPDH (ab8245), and nonspecific rabbit IgG isotype control (ab37415) were purchased from Abcam (Cambridge, MA, USA). The antibody against NM1 has been previously characterized2 (link). Alexa Fluor 555 Goat Anti-Rabbit (ab150078) and Alexa Fluor 488 Goat Anti-Mouse (ab150117) were used as secondary antibodies for immunofluorescence, and horseradish peroxidase (HRP)-fused Goat Anti-Rabbit (ab6721) and Rabbit Anti-Mouse (ab6728) secondary antibodies for western blottings were purchased from Abcam. Hoechst 43222 (H1399) and ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI; P36931) were purchased from Invitrogen, Waltham, MA, USA. All antibodies have been used according to the manufacturers’ protocols.
Initial experiments were performed on primary MEFs derived from 13.5 days NM1 WT and KO embryos46 (link). In other experiments were used immortalized MEFs (ATCC® CRL-2752) or cell lines derived from these immortalized cells.
The antibodies against H3K9me3 (ab8898), H3K27ac (ab4729), H3K4me1 (ab8895), H3K4me3 (ab8580), H3K9ac (ab10812), H3 (ab1791), γH2AX (ab2893), p53-K370 (ab183544), PCAF (ab12188), Set1 (ab70378), GAPDH (ab8245), and nonspecific rabbit IgG isotype control (ab37415) were purchased from Abcam (Cambridge, MA, USA). The antibody against NM1 has been previously characterized2 (link). Alexa Fluor 555 Goat Anti-Rabbit (ab150078) and Alexa Fluor 488 Goat Anti-Mouse (ab150117) were used as secondary antibodies for immunofluorescence, and horseradish peroxidase (HRP)-fused Goat Anti-Rabbit (ab6721) and Rabbit Anti-Mouse (ab6728) secondary antibodies for western blottings were purchased from Abcam. Hoechst 43222 (H1399) and ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI; P36931) were purchased from Invitrogen, Waltham, MA, USA. All antibodies have been used according to the manufacturers’ protocols.
4
Western Blot Analysis of DNA Damage Response
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Whole-cell protein lysates of treated cells were extracted and fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose as previously described 14 (link). Protein expression was determined using the Western Lightning Plus-ECL detection system (NEL104001EA, PerkinElmer, MA, USA). Primary antibodies used: γ-H2AX (ab2893, abcam), activated caspase3 (ab32042, abcam), CHK2 pThr 68 (2661, Cell Signaling), CHK2 (3440, Cell Signaling), ATM pSer 1981 (5883, Cell Signaling) and GAPDH (MAB374, Millipore). Secondary antibodies used: anti-rabbit IgG, HRP-linked antibody (7074, Cell Signaling) and anti-mouse IgG, HRP-linked antibody (7076, Cell Signaling).
5
Comprehensive Immunoblotting Antibody Panel
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The following antibodies were used: rabbit polyclonal antibody against phospho S139 gamma H2A.X (ab2893, Abcam), rabbit monoclonal antibody against alpha-tubulin (ab52866, Abcam), mouse monoclonal antibody against UBF (sc13125, Santa Cruz), RPA194 (sc48385, Santa Cruz), phospho S139 gamma H2A.X antibody (ab26350, Abcam), Anti-BrdU antibody (B8434, Sigma), sheep polyclonal antibody against BrdU (ab1893, Abcam), Alexa 488-conjugated goat anti-mouse IgG (A11029, Invitrogen), Alexa 568-conjugated goat anti-rabbit IgG (A11011, Invitrogen), DyLight 488 goat anti-mouse IgG (ab96879, Abcam), isotype mouse IgG antibody (ab91353, Abcam), alkaline phosphate-conjugated rabbit IgG (ZB5305, ZSGB) and HRP goat anti-mouse IgG (A0208, Beyotime).
6
Mitochondrial and Nuclear Protein Extraction
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The antibodies for ERRα (ab37438), H2AX (ab20669), γH2AX (ab2893), H3K9me2 (ab1220), H3K9me3 (ab8898) and H3K27me3 (ab6002) were obtained from Abcam, and all the other antibodies, including β-actin (sc-47778), eNOS (sc-654), ERβ (sc-137381) and SOD2 (sc-30080) were obtained from Santa Cruz Biotechnology. The mitochondrial fraction was isolated using a Pierce Mitochondria Isolation Kit (Pierce Biotechnology) according to manufacturers’ instructions. Nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce Biotechnology). Protein concentration was measured using the Coomassie Protein Assay Kit (Pierce Biotechnology) according to manufacturers’ instructions.
7
EV Effects on Cell Proliferation and DNA Damage
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To evaluate the effects of EVs on cell proliferation and DNA damage, HUVEC (4×104) and H9c2 cells (4×104) were seeded on 4-well chamber culture slides. After 72 h of culture with 10 μg/ml non-irradiated-EVs or irradiated-EVs (n=3), the cells were washed with PBS and fixed in 4% paraformaldehyde for 10 min. After the incubation of protein block serum-free (DAKO) with 0.01% Triton X-100, the cells were incubated with Ki67 monoclonal antibody (SolA15, Invitrogen), anti-53BP1 antibody (ab36823, Abcam), or anti-gamma H2A.X (ab2893, Abcam), followed by associated Alexa flour 488-conjugated second antibody. Nuclei were stained with DAPI, the positively stained cells were counted under fluorescence microscopy at 200-fold magnification, and 20 fields per section were randomly selected for quantitative counting. The percentage of positive cells in each field was calculated as (positively stained cells/all cells in the field)×100%.
8
DNA Damage Response Evaluation
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LoVo and SW480 cells were treated with 20 μM SSa for 15 h in the presence or absence of z-LEVD-fmk (caspase-4 inhibitor), then fixed in 4% paraformaldehyde at 4°C for 15 min. The fixed cells were permeabilized in PBS containing 100 mM Tris-HCl, 50 mM ethylenediamine tetra-acetic acid (EDTA), and 0.5% Triton X-100 for 15 min at room temperature. Cells were incubated with phospho-H2AX (ab2893; Abcam, Cambridge, UK) primary antibody diluted to a 1:100 ratio at 4°C overnight and then detected with AlexaFluor 594-conjugated anti-rabbit secondary antibody (A-11037; Molecular Probes, Eugene, OR, USA). Slides were counterstained with DAPI, and images were acquired on an ECLIPSE E600 fluorescence microscope (Nikon, Tokyo, Japan). Cells were classified as ‘positive’ when more than three H2AX foci were observed per cell.
9
Sirt1 Inhibitor Screening in MEFs
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Sirt1 WT and KO MEFS cells were treated with 10 μM EX-527, 12 μM S1th 13 (6 ), 20 μM S1th 12 (5 ), 30 μM S1th 10 (7 ) and 30 μM SirReal2 [Sirt2 inhibitor, synthesized according to (62 (link))] for 48 h. Oxidative stress was induced using 10 μM Camptothecin 2 h. Whole-cell extracts were performed according to the Dignam protocol (63 (link)). Primary antibodies used for the western blot were anti-H2AX and anti-γH2AX (ab11175 and ab2893 resp., Abcam, UK). Densiometric analysis of the western blots was performed with Quantity One software (Bio-Rad Laboratories, Inc., USA).
10
Immunofluorescent Staining of Cell Lines
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The cell lines were plated onto cover-slips and treated, respectively. After 48 h, cells were fixed with 4% paraformaldehyde for 10 min at room temperature. All slides were then washed with PBS and blocked with Goat serum at room temperature for 30 min. Cells were incubated with primary antibodies (γH2AX: Abcam, ab2893, 1:400; CD206: Abcam, ab64693, 1:400) at 4 °C overnight, washed with PBS, and then incubated with appropriated secondary antibodies for 1 h at room temperature. Cells were examined immediately using Nikon C-HGFI Intensilight Fiber Illuminator (Nikon, Japan) fluorescence microscope.
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