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Phospho-Histone H2A.X (S139) Recombinant Rabbit Monoclonal Antibody [SR33-09]

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Specification

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Catalog# ET1602-2

Phospho-Histone H2A.X (S139) Recombinant Rabbit Monoclonal Antibody [SR33-09]

Application

  • WB

  • IHC-P

  • IF-Cell

  • IF-Tissue

Reactivity

  • Human

  • Mouse

  • Rat

  • Cynomolgus monkey

BSA and Azide free
Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

Phospho-Histone H2A.X (S139) Recombinant Rabbit Monoclonal Antibody [SR33-09]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding Ser139 of Human Histone H2AX.

Species Reactivity

Human, Mouse, Rat, Cynomolgus monkey

Validated Applications

WB, IHC-P, IF-Cell, IF-Tissue

Molecular Weight

Predicted band size: 15 kDa

Positive Control

NIH/3T3 cell lysate, NIH/3T3 treated with 25μM Etoposide for 5 hours cell lysate, HeLa treated with 20μM Etoposide for 2 hours whole cell lysate, HeLa treated with UV for 2 hours whole cell lysate, C6 treated with 25μM Etoposide for 5 hours whole cell lysate, rat brain tissue, mouse testis tissue, mouse small intestine tissue, human colon carcinoma tissue, human colon cancer tissue, mouse breast tissue, mouse skin tissue, HeLa cells treated with 20μM etoposide for 2 hours.

Conjugation

unconjugated

Clone Number

SR33-09

RRID

AB_3069632

Product Features

Form

Liquid

Concentration

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). Store at -20℃ long term.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:2,000-1:5,000

  • IHC-P

  • 1:500-1:5,000

  • IF-Cell

  • 1:2,000-1:3,000

  • IF-Tissue

  • 1:500

Target

Function

Histone H2A.X is a member of the Histone H2A family, which is involved in nucleosomal organization of chromatin. An important phosphorylated form is γH2AX (S139), which forms when double-strand breaks appear. In humans and other eukaryotes, the DNA is wrapped around histone octamers, consisting of core histones H2A, H2B, H3 and H4, to form chromatin. H2AX contributes to nucleosome-formation, chromatin-remodeling and DNA repair, and is also used in vitro as an assay for double-strand breaks in dsDNA.

Background References

1. Kung, M.L. et al. 2015. Enhanced reactive oxygen species overexpression by CuO nanoparticles in poorly differentiated hepatocellular carcinoma cells. Nanoscale. 7: 1820-9.

2. Cilli, D. et al. 2014. Identification of the interactors of human nibrin (NBN) and of its 26 kDa and 70 kDa fragments arising from the NBN 657del5 founder mutation. PloS one. 9: e114651.

Sequence Similarity

Belongs to the histone H2A family.

Post-translational Modification

Phosphorylated on Ser-140 (to form gamma-H2AX or H2AX139ph) in response to DNA double strand breaks (DSBs) generated by exogenous genotoxic agents and by stalled replication forks, and may also occur during meiotic recombination events and immunoglobulin class switching in lymphocytes. Phosphorylation can extend up to several thousand nucleosomes from the actual site of the DSB and may mark the surrounding chromatin for recruitment of proteins required for DNA damage signaling and repair. Widespread phosphorylation may also serve to amplify the damage signal or aid repair of persistent lesions. Phosphorylation of Ser-140 (H2AX139ph) in response to ionizing radiation is mediated by both ATM and PRKDC while defects in DNA replication induce Ser-140 phosphorylation (H2AX139ph) subsequent to activation of ATR and PRKDC. Dephosphorylation of Ser-140 by PP2A is required for DNA DSB repair. In meiosis, Ser-140 phosphorylation (H2AX139ph) may occur at synaptonemal complexes during leptotene as an ATM-dependent response to the formation of programmed DSBs by SPO11. Ser-140 phosphorylation (H2AX139ph) may subsequently occurs at unsynapsed regions of both autosomes and the XY bivalent during zygotene, downstream of ATR and BRCA1 activation. Ser-140 phosphorylation (H2AX139ph) may also be required for transcriptional repression of unsynapsed chromatin and meiotic sex chromosome inactivation (MSCI), whereby the X and Y chromosomes condense in pachytene to form the heterochromatic XY-body. During immunoglobulin class switch recombination in lymphocytes, Ser-140 phosphorylation (H2AX139ph) may occur at sites of DNA-recombination subsequent to activation of the activation-induced cytidine deaminase AICDA. Phosphorylation at Tyr-143 (H2AXY142ph) by BAZ1B/WSTF determines the relative recruitment of either DNA repair or pro-apoptotic factors. Phosphorylation at Tyr-143 (H2AXY142ph) favors the recruitment of APBB1/FE65 and pro-apoptosis factors such as MAPK8/JNK1, triggering apoptosis. In contrast, dephosphorylation of Tyr-143 by EYA proteins (EYA1, EYA2, EYA3 or EYA4) favors the recruitment of MDC1-containing DNA repair complexes to the tail of phosphorylated Ser-140 (H2AX139ph).; Monoubiquitination of Lys-120 (H2AXK119ub) by RING1 and RNF2/RING2 complex gives a specific tag for epigenetic transcriptional repression (By similarity). Following DNA double-strand breaks (DSBs), it is ubiquitinated through 'Lys-63' linkage of ubiquitin moieties by the E2 ligase UBE2N and the E3 ligases RNF8 and RNF168, leading to the recruitment of repair proteins to sites of DNA damage. Ubiquitination at Lys-14 and Lys-16 (H2AK13Ub and H2AK15Ub, respectively) in response to DNA damage is initiated by RNF168 that mediates monoubiquitination at these 2 sites, and 'Lys-63'-linked ubiquitin are then conjugated to monoubiquitin; RNF8 is able to extend 'Lys-63'-linked ubiquitin chains in vitro. H2AK119Ub and ionizing radiation-induced 'Lys-63'-linked ubiquitination (H2AK13Ub and H2AK15Ub) are distinct events.; Acetylation at Lys-37 increases in S and G2 phases. This modification has been proposed to play a role in DNA double-strand break repair (By similarity).

Subcellular Location

Nucleus, Chromosome

UNIPROT

SwissProt: P16104 Human

SwissProt: P27661 Mouse

Unigene: 2850 Rat

Synonyms

AW228881 antibody

H2A histone family member X antibody

H2A.FX antibody

H2A.X antibody

H2a/x antibody

H2AFX antibody

H2AX antibody

H2AX histone antibody

H2AX_HUMAN antibody

Hist5.2ax antibody

Expand

Images

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-Histone H2A.X (S139) on different lysates with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/5,000 dilution.<br /><br />Lane 1: NIH/3T3 cell lysate (20 µg/Lane)<br />Lane 2: NIH/3T3 treated with 25μM Etoposide for 5 hours cell lysate (20 µg/Lane)<br /><br />Predicted band size: 15 kDa<br />Observed band size: 15 kDa<br /><br />Exposure time: 6 seconds; ECL: K1801;<br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    ☑ Cell treatment (CT)

    Western blot analysis of Phospho-Histone H2A.X (S139) on different lysates with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/5,000 dilution.

    Lane 1: NIH/3T3 cell lysate (20 µg/Lane)
    Lane 2: NIH/3T3 treated with 25μM Etoposide for 5 hours cell lysate (20 µg/Lane)

    Predicted band size: 15 kDa
    Observed band size: 15 kDa

    Exposure time: 6 seconds; ECL: K1801;
    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1602-2) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-Histone H2A.X (S139) on different lysates with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/2,000 dilution.<br /><br />Lane 1: HeLa whole cell lysate<br />Lane 2: HeLa treated with 20μM Etoposide for 2 hours whole cell lysate<br />Lane 3: HeLa whole cell lysate<br />Lane 4: HeLa treated with UV for 2 hours whole cell lysate<br />Lane 5: NIH/3T3 whole cell lysate<br />Lane 6: NIH/3T3 treated with 25μM Etoposide for 5 hours whole cell lysate<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 15 kDa<br />Observed band size: 15/20 kDa<br /><br />Exposure time: 53 seconds;<br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/2,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/100,000 dilution was used for 1 hour at room temperature.

    ☑ Cell treatment (CT)

    Western blot analysis of Phospho-Histone H2A.X (S139) on different lysates with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/2,000 dilution.

    Lane 1: HeLa whole cell lysate
    Lane 2: HeLa treated with 20μM Etoposide for 2 hours whole cell lysate
    Lane 3: HeLa whole cell lysate
    Lane 4: HeLa treated with UV for 2 hours whole cell lysate
    Lane 5: NIH/3T3 whole cell lysate
    Lane 6: NIH/3T3 treated with 25μM Etoposide for 5 hours whole cell lysate

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 15 kDa
    Observed band size: 15/20 kDa

    Exposure time: 53 seconds;
    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1602-2) at 1/2,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/100,000 dilution was used for 1 hour at room temperature.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-Histone H2A.X (S139) on different lysates with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/2,000 dilution.<br /><br />Lane 1: C6 treated with 25μM Etoposide for 5 hours whole cell lysate<br />Lane 2: C6 whole cell lysate<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 15 kDa<br />Observed band size: 15 kDa<br /><br />Exposure time: 20 seconds; ECL: K1801;<br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    ☑ Cell treatment (CT)

    Western blot analysis of Phospho-Histone H2A.X (S139) on different lysates with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/2,000 dilution.

    Lane 1: C6 treated with 25μM Etoposide for 5 hours whole cell lysate
    Lane 2: C6 whole cell lysate

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 15 kDa
    Observed band size: 15 kDa

    Exposure time: 20 seconds; ECL: K1801;
    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1602-2) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/500 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/500 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1602-2) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded mouse testis tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/500 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded mouse testis tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/500 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1602-2) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded mouse small intestine tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/500 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded mouse small intestine tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/500 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1602-2) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/500 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/500 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1602-2) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded human colon cancer tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/500 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded human colon cancer tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/500 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1602-2) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded mouse breast tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/5,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/5,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded mouse breast tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/5,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1602-2) at 1/5,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded mouse skin tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/500 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded mouse skin tissue with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/500 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1602-2) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Immunocytochemistry analysis of HeLa cells untreated / treated with 20μM etoposide for 2 hours labeling Phospho-Histone H2A.X (S139) with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/2,000 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/2,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />Beta tubulin (<a href="/products/HA601187" style="font-weight: bold;text-decoration: underline;">HA601187</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    ☑ Cell treatment (CT)

    Immunocytochemistry analysis of HeLa cells untreated / treated with 20μM etoposide for 2 hours labeling Phospho-Histone H2A.X (S139) with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/2,000 dilution.

    Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/2,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

  • Immunocytochemistry analysis of 4T1 cells labeling Phospho-Histone H2A.X (S139) with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/3,000 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (<a href="/products/ET1602-2" style="font-weight: bold;text-decoration: underline;">ET1602-2</a>) at 1/3,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />Beta tubulin (<a href="/products/HA601187" style="font-weight: bold;text-decoration: underline;">HA601187</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of 4T1 cells labeling Phospho-Histone H2A.X (S139) with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/3,000 dilution.

    Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-Histone H2A.X (S139) antibody (ET1602-2) at 1/3,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

  • Application: IF-tissue<br /><br />Species: Cynomolgus monkey<br /><br />Site: Lung <br /><br />Sample: Paraffin-embedded section<br /><br />Antibody concentration: 1/500

    Application: IF-tissue

    Species: Cynomolgus monkey

    Site: Lung

    Sample: Paraffin-embedded section

    Antibody concentration: 1/500

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