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  1. Link

    histone-h2ax-antibody-chip-grade-ab20669.pdf

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Epigenetics and Nuclear Signaling Histones Variants
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Validated using a knockout cell line

Anti-Histone H2A.X antibody - ChIP Grade (ab20669)

  • Datasheet
  • SDS
Reviews (7) Submit a question References (27)

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Western blot - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
  • Western blot - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
  • ChIP - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
  • Immunoprecipitation - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
  • Western blot - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)

Key features and details

  • Rabbit polyclonal to Histone H2A.X - ChIP Grade
  • Suitable for: ChIP, WB, IP, IHC-P
  • Knockout validated
  • Reacts with: Mouse, Rat, Human, Recombinant fragment
  • Isotype: IgG

Get better batch-to-batch reproducibility with a recombinant antibody

Product image
Anti-Histone H2A.X antibody [EPR895] (ab124781)
  • Research with confidence – consistent and reproducible results with every batch
  • Long-term and scalable supply – powered by recombinant technology for fast production
  • Success from the first experiment – confirmed specificity through extensive validation
  • Ethical standards compliant – production is animal-free

Overview

  • Product name

    Anti-Histone H2A.X antibody - ChIP Grade
    See all Histone H2A.X primary antibodies
  • Description

    Rabbit polyclonal to Histone H2A.X - ChIP Grade
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ChIP, WB, IP, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human, Recombinant fragment
  • Immunogen

    Synthetic peptide corresponding to Human Histone H2A.X aa 100 to the C-terminus (C terminal) conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab15646)

  • General notes

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituent: PBS

    Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
  • Concentration information loading...
  • Purity

    Immunogen affinity purified
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

    • Epigenetics and Nuclear Signaling
    • Histones
    • Variants
    • Epigenetics and Nuclear Signaling
    • DNA / RNA
    • DNA Damage & Repair
    • DNA Damage Response
    • DNA Damage Recognition
    • Epigenetics and Nuclear Signaling
    • Histones
    • H2A
    • Unmodified
    • Epigenetics and Nuclear Signaling
    • ChIP assays
    • ChIP antibodies

Associated products

  • ChIP Related Products

    • ChIP Kit (ab500)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
    •  Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776)
  • Control Peptide

    • Human gamma H2A.X (phospho S139) peptide (ab15645)
  • Isotype control

    • Rabbit IgG, polyclonal - Isotype Control (ChIP Grade) (ab171870)
  • Recombinant Protein

    • Recombinant Human Histone H2A.X protein (ab134863)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab20669 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ChIP
Use 2 µg for 25 µg of chromatin.
WB (3)
Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).
IP
Use at an assay dependent concentration.
IHC-P
Use a concentration of 0.2 - 0.5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Notes
ChIP
Use 2 µg for 25 µg of chromatin.
WB
Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).
IP
Use at an assay dependent concentration.
IHC-P
Use a concentration of 0.2 - 0.5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Target

  • Function

    Variant histone H2A which replaces conventional H2A in a subset of nucleosomes. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling. Required for checkpoint-mediated arrest of cell cycle progression in response to low doses of ionizing radiation and for efficient repair of DNA double strand breaks (DSBs) specifically when modified by C-terminal phosphorylation.
  • Sequence similarities

    Belongs to the histone H2A family.
  • Developmental stage

    Synthesized in G1 as well as in S-phase.
  • Domain

    The [ST]-Q motif constitutes a recognition sequence for kinases from the PI3/PI4-kinase family.
  • Post-translational
    modifications

    Phosphorylated on Ser-140 (to form gamma-H2AFX 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. 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. Monoubiquitination and ionizing radiation-induced 'Lys-63'-linked ubiquitination are distinct events.
  • Cellular localization

    Nucleus. Chromosome.
  • Target information above from: UniProt accession P16104 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 3014 Human
    • Entrez Gene: 15270 Mouse
    • Entrez Gene: 500987 Rat
    • Omim: 601772 Human
    • SwissProt: P16104 Human
    • SwissProt: P27661 Mouse
    • Unigene: 477879 Human
    • Unigene: 245931 Mouse
    • Alternative names

      • 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
      • Histone 2A antibody
      • Histone 2AX antibody
      • Histone H2A.X antibody
      • Histone H2AX antibody
      • RGD1566119 antibody
      see all

    Images

    • Western blot - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
      Western blot - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
      All lanes : Anti-Histone H2A.X antibody - ChIP Grade (ab20669) at 1 µg/ml

      Lane 1 : Wild-type HAP1 whole cell lysate
      Lane 2 : H2AFX (Histone H2A.X) knockout HAP1 whole cell lysate
      Lane 3 : HEK 293 whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Predicted band size: 15 kDa



      Lanes 1 - 3: Merged signal (red and green). Green - ab20669 observed at 17 kDa. Red - loading control, ab9484, observed at 37 kDa.

      ab20669 was shown to recognize Histone H2A.X in wild-type HAP1 cells as signal was lost at the expected MW in H2AFX (Histone H2A.X) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and H2AFX (Histone H2A.X) knockout samples were subjected to SDS-PAGE. Ab20669 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

    • Western blot - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
      Western blot - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
      All lanes : Anti-Histone H2A.X antibody - ChIP Grade (ab20669) at 1 µg/ml

      Lane 1 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 10 µg
      Lane 2 : HeLa Histone Preparation Nuclear Lysate at 2.5 µg
      Lane 3 : Histone H2A Recombinant Protein (negative control) at 0.1 µg

      Secondary
      All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution

      Developed using the ECL technique.

      Performed under reducing conditions.

      Predicted band size: 15 kDa
      Observed band size: 17 kDa why is the actual band size different from the predicted?


      Exposure time: 20 minutes
    • ChIP - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
      ChIP - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)

      Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab20669 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)

      IHC image of ab20669 staining Histone H2A.X in human colon formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab20669, 0.5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the negative control (shown on the inset).

       

      For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    • Immunocytochemistry/ Immunofluorescence - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
      Immunocytochemistry/ Immunofluorescence - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)This image is courtesy of an anonymous Abreview

      ab20669 staining Histone H2A.X in Mouse brain neurone cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.3% Triton X-100 and blocked with 5% serum for 2 hours at 22°C. Samples were incubated with primary antibody (1/500 in 2.5% serum + 0.3% Triton X-100 in PBS) for 24 hours at 4°C. ab175651, an Alexa Fluor® 405-conjugated Donkey anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

      See Abreview

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)

      IHC image of Histone H2A.X staining in Human Lung formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab20669, 0.2 µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

       

      For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    • Immunocytochemistry/ Immunofluorescence - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
      Immunocytochemistry/ Immunofluorescence - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)This image is courtesy of an Abreview submitted by Dr Kirk McManus
      ab20669 (1/2000) staining Histone H2A.X in Hela cells (green). Cells were fixed in methanol and counter-stained with DAPI in order to highlight the nucleus (red). Please refer to abreview for further experimental details.

      See Abreview

    • Immunoprecipitation - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
      Immunoprecipitation - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
      Histone H2A.X was immunoprecipitated using 0.5mg Rat Testis whole tissue extract, 5µg of Rabbit polyclonal to Histone H2A.X and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
      The antibody was incubated under agitation with Protein G beads for 10min, Rat Testis whole tissue extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
      Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab20669.
      Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
      Band: 17kDa: Histone H2A.X.
    • Western blot - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
      Western blot - Anti-Histone H2A.X antibody - ChIP Grade (ab20669)
      All lanes : Anti-Histone H2A.X antibody - ChIP Grade (ab20669) at 1 µg/ml

      Lane 1 : Lung (Mouse) Tissue Lysate
      Lane 2 : Testis (Mouse) Tissue Lysate
      Lane 3 : Lung (Rat) Tissue Lysate
      Lane 4 : Testis (Rat) Tissue Lysate - normal tissue (ab29388)

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

      Developed using the ECL technique.

      Performed under reducing conditions.

      Predicted band size: 15 kDa
      Observed band size: 17 kDa why is the actual band size different from the predicted?
      Additional bands at: 55 kDa. We are unsure as to the identity of these extra bands.


      Exposure time: 8 minutes

    Protocols

    • ChIP protocols
    • Immunoprecipitation protocols
    • Immunohistochemistry protocols
    • Western blot protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (27)

    Publishing research using ab20669? Please let us know so that we can cite the reference in this datasheet.

    ab20669 has been referenced in 27 publications.

    • Rübe CE  et al. Human skin aging is associated with increased expression of the histone variant H2A.J in the epidermis. NPJ Aging Mech Dis 7:7 (2021). PubMed: 33795696
    • Xie W  et al. Pterostilbene accelerates wound healing by modulating diabetes-induced estrogen receptor ß suppression in hematopoietic stem cells. Burns Trauma 9:tkaa045 (2021). PubMed: 33654697
    • Xu X  et al. The epigenetic regulator LSH maintains fork protection and genomic stability via MacroH2A deposition and RAD51 filament formation. Nat Commun 12:3520 (2021). PubMed: 34112784
    • McMahon KA  et al. Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response. Elife 10:N/A (2021). PubMed: 34142659
    • Gao X  et al. Systematic analysis of lysine acetylome and succinylome reveals the correlation between modification of H2A.X complexes and DNA damage response in breast cancer. Oncol Rep 43:1819-1830 (2020). PubMed: 32236595
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-7 of 7 Abreviews or Q&A

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Histone H2A.X antibody - ChIP Grade

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (normal fibroblasts)
    Permeabilization
    Yes - 0.1% Triton X-100 in TBS for 10 minutes
    Specification
    normal fibroblasts
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 23°C
    Fixative
    Paraformaldehyde
    Read More

    Abcam user community

    Verified customer

    Submitted Dec 11 2017

    Western blot abreview for Anti-Histone H2A.X antibody - ChIP Grade

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (breast cancer)
    Gel Running Conditions
    Non-reduced Denaturing (gel 10%)
    Loading amount
    40 µg
    Specification
    breast cancer
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
    Read More

    Miss. fan zhang

    Verified customer

    Submitted Apr 19 2017

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Histone H2A.X antibody

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Blocking step
    Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
    Sample
    Mouse Cell (Brain, neurons)
    Specification
    Brain, neurons
    Permeabilization
    Yes - 0.3% Triton
    Fixative
    Paraformaldehyde
    Read More

    Abcam user community

    Verified customer

    Submitted Dec 19 2013

    Western blot abreview for Anti-Histone H2A.X antibody

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Loading amount
    10 µg
    Gel Running Conditions
    Reduced Denaturing (4-12% gradient gel)
    Sample
    Human Cell lysate - other (lymphocytes)
    Specification
    lymphocytes
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 37°C
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 21 2013

    Western blot abreview for Anti-Histone H2A.X antibody

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (293T cell)
    Loading amount
    30 µg
    Specification
    293T cell
    Gel Running Conditions
    Reduced Denaturing (4-12% Gradient Gel)
    Blocking step
    Licor Blocking Buffer as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 10 2009

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Histone H2A.X antibody

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa)
    Specification
    HeLa
    Fixative
    Methanol
    Permeabilization
    No
    Read More

    DR. Kirk Mcmanus

    Verified customer

    Submitted Oct 13 2008

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Histone H2A.X antibody

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Mouse Cell (10T1/2)
    Specification
    10T1/2
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.5% Triton X100 in PBS
    Read More

    DR. Kirk Mcmanus

    Verified customer

    Submitted Sep 12 2008

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