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  1. Link

    rabbit-specific-hrpdab-abc-detection-ihc-kit-ab64261.pdf

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Rabbit specific HRP/DAB (ABC) Detection IHC Kit (ab64261)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (3)Q&A (13)References (45)

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Immunohistochemistry - ab64261
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti Rabbit HRP/DAB Detection Kit (ab64261)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti Rabbit HRP/DAB Detection Kit (ab64261)

Overview

  • Product name

    Rabbit specific HRP/DAB (ABC) Detection IHC Kit
    See all HRP/DAB detection kit kits
  • Product overview

    Rabbit specific HRP/DAB (ABC) IHC Detection Kit ab64261 is a complete kit to detect a primary antibody raised in rabbit, using HRP-labeled-streptavidin and a biotinylated anti-rabbit secondary antibody.


    It is supplied with a full protocol and with peroxidase and protein blocking reagents, biotinylated secondary antibody, streptavidin-HRP, and DAB substrate.


    In the method:
    - a sample is incubated with an unconjugated primary antibody specific to the target antigen
    - a biotinylated secondary antibody then binds to the primary antibody
    - HRP-labeled streptavidin then binds to the secondary antibody
    - the HRP produces a brown colored substance at the site of primary antibody binding by reacting with DAB.

  • Notes

    For alternative streptavidin-biotin IHC detection kits see:
    - Anti-Mouse / anti-Rabbit HRP-DAB IHC kit ab64264
    - Anti-Mouse HRP-DAB IHC Kit ab64259

    For high sensitivity HRP micro-polymer IHC detection kits see:
    - Anti-Mouse / anti-Rabbit HRP/DAB Micro-polymer IHC Kit ab236466
    - Anti-Rabbit HRP-DAB Micro-polymer IHC Kit ab236469

    Alternatively, see HRP-AEC IHC detection kits.

    Find more kits and reagents for antigen retrieval, blocking, signal amplification, visualization, counterstaining, and mounting in the IHC kits and reagents guide.

  • Tested applications

    Suitable for: IHC-P, IHC-Frmore details

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components 15 ml
    50x DAB Chromogen 1 x 0.5ml
    Biotinylated goat anti-rabbit IgG(H+L) 1 x 15ml
    DAB substrate 1 x 15ml
    Hydrogen Peroxide Block 1 x 15ml
    Protein block 1 x 15ml
    Streptavidin Peroxidase 1 x 15ml
  • Research areas

    • Kits/ Lysates/ Other
    • Kits
    • IHC Kits
    • Kits/ Lysates/ Other
    • Kits
    • IHC tools
    • ABC
  • Relevance

    HRP/DAB detection kit is a immunoenzymatic antigen detection system for immunohistochemistry techniques.

Applications

Our Abpromise guarantee covers the use of ab64261 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
IHC-P Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.

Images

  • Immunohistochemistry - ab64261
    Immunohistochemistry - ab64261Image from Narendra S.C., PLoS One 10(10), Fig 5a. doi: 10.1371/journal.pone.0141863 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    Immunohistochemical analysis staining TNF or IL6 in mouse tissue sections.  Samples are incubated in murine TNF (ab9739) or IL6 primary antibody. Bound primary antibodies were detected using IHC detection kit (ab64261). Immunohistochemistry was used to determine the effect of uridine on synovial expression of TNF and IL6. 

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti Rabbit HRP/DAB Detection Kit (ab64261)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti Rabbit HRP/DAB Detection Kit (ab64261)This image is courtesy of an anonymous Abreview

    Immunohistochemical analysis of KLF4 and pVHL expression in colon cancer tissues. Sections from colon cancer and adjacent tissues were analyzed by immunostaining against KLF4 and pVHL. Staining without primary antibody served as negative control.

    Tissue sections were de-waxed with xylene and rehydrated through gradient ethanol into water. For antigen retrieval, sections were heated in citrate buffer (pH6.0) for 10 min at 95°C in a microwave oven. After cooling to room temperature, the sections were then digested with 0.05% trypsin for 10 min at 37°C. Endogenous peroxidase activity was quenched with 0.3% H2O2 in methanol for 30 min at room temperature. After PBS washes, nonspecific antibody binding was blocked by pre-incubating slides with 10% normal goat non-immune serum at 37° for 30 min. After blotting off the blocking serum, sections were incubated with primary antibody against LKF4 as well as primary antibody against VHL at 4° overnight. After PBS washes again, sections were incubated with biotinylated secondary antibody at 1:200 dilution for 30 min at room temperature. After incubating with Vectastain ABC reagent (Vector Laboratories, Inc., Burlingame, CA) for 30 min at room temperature, the sections were developed with Anti Rabbit HRP/DAB Detection Kit (ab64261). Sections were washed in running tap water and lightly counterstained with hematoxylin, followed by dehydration and coverslip mounting. Negative controls were obtained by omitting the primary antibody.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti Rabbit HRP/DAB Detection Kit (ab64261)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti Rabbit HRP/DAB Detection Kit (ab64261)
    Human colon carcinoma fixed in 10% NBF for 24 hrs and stained with anti p53 antibody using Anti Rabbit HRP/DAB Detection Kit (ab64261).

Protocols

  • Rabbit specific HRP/DAB detection kit
  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

    • Datasheet
    • SDS
  • References (45)

    Publishing research using ab64261? Please let us know so that we can cite the reference in this datasheet.

    ab64261 has been referenced in 45 publications.

    • Jung YJ  et al. Cell reprogramming using extracellular vesicles from differentiating stem cells into white/beige adipocytes. Sci Adv 6:eaay6721 (2020). PubMed: 32232152
    • Shinde T  et al. Synbiotic Supplementation Containing Whole Plant Sugar Cane Fibre and Probiotic Spores Potentiates Protective Synergistic Effects in Mouse Model of IBD. Nutrients 11:N/A (2019). PubMed: 30979002
    • Badran M  et al. Intermittent hypoxia impairs uterine artery function in pregnant mice. J Physiol 597:2639-2650 (2019). PubMed: 31002746
    • Saiz AM  et al. Conditioning of myoblast secretome using mesenchymal stem/stromal cell spheroids improves bone repair. Bone 125:151-159 (2019). PubMed: 31102712
    • Shao L  et al. Activating metabotropic glutamate receptor-7 attenuates visceral hypersensitivity in neonatal maternally separated rats. Int J Mol Med 43:761-770 (2019). PubMed: 30569115
    View all Publications for this product

    Customer reviews and Q&As

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

    1-10 of 16 Abreviews or Q&A

    Question

    I have been using the DAB staining kit for my IHC, and it seems to work well, except that the results are somewhat "patchy" across the slide. For example, the sections on the bottom half of the slide stain much more strongly than those on the top half, and individual sections may have distinct areas of light and dark staining not associated with differences in expression. I see no staining on my no primary controls. I have tried to make sure that the reagents cover the slide evenly. I thought that uneven antigen retrieval might be a problem (I use a microwave method), but I have done a similar AR using immunoflourescence and didn't notice these discrepancies. Do you have any suggestions?

    Read More

    Abcam community

    Verified customer

    Asked on Mar 18 2013

    Answer

    This issue may be result of sample preparation. It is oftentimes the case that the slides have not been inadequate deparaffinization which will lead to this type of staining. The link below outlines Abcam recommendations of deparaffinization: https://www.abcam.com/index.html?pageconfig=resource&rid=11487 Another cause can be insufficient fixation. If formalin has not had sufficient time to penetrate and fix the tissue, there will be uneven fixation, and as a result uneven staining.

    Read More

    Abcam Scientific Support

    Answered on Mar 18 2013

    Question

    The kit came with dropper bottles - how does this work? And I don't need a whole ml of DAB, so can I store it once I've added the chromogen? Or can I scale it down?

    Read More

    Abcam community

    Verified customer

    Asked on Mar 05 2013

    Answer

    One drop of DAB chromogen is about 30 ul, and it is to be added to 1.5 ml of the substrate (50 drops). So the ratio is 1:50.

    The mixture cannot be stored very long once you have made it. Ideally, you should mix it immediately before using it, and store for no more than an hour.

    You can scale down but it will require either pulling off the dropper part from the bottles each time in order to pipet the required quantity (for example, 10 ul of chromogen and 500 ul of substrate). It will be better to transfer the contents of each reagent to a new container (for example, an Eppendorf tube wrapped in foil to protect from light for the chromogen and a 15ml tube for the substrate).

    Read More

    Abcam Scientific Support

    Answered on Mar 05 2013

    Question

    Dear Sirs,
    could you please confirm that the protein block included in the Rabbit specific HRP/DAB (ABC) Detection IHC Kit (code# ab64261) is made of the following components: 0.5% BSA, 0.5% Casein, PBS
    Thank you in advance,

    Read More

    Abcam community

    Verified customer

    Asked on Jan 10 2013

    Answer


    I can confirm the Protein Block is a PBS solution with 0.5% Casein and 0.5% BSA.

    I hope this information will be helpful for your customer.
    Have a great day!

    Read More

    Abcam Scientific Support

    Answered on Jan 10 2013

    Great product that gives best results each time.

    Excellent Excellent 5/5 (Ease of Use)
    Abreviews
    Abreviews
    abreview image
    We are very happy using this kit. Every time without fail we get strong signals. perfectly drafted protocol for easy use. Needs optimization with time of incubation depending on the immunoreactivity and intensity of your primary antibody.

    Abcam user community

    Verified customer

    Submitted May 08 2018

    Question

    I have bought the ab64261 kit. The protocol is for paraffin-embedded slides. I wondered if you have a protocol for frozen sections. For example
    Do they have to be fixed? If so with what? Acetone?
    - Do I have to carry the hydrogen peroxide block step before or after fixing?
    - Do I have to dilute my primary in the Protein block provided?
    - What Washing buffer do you suggest? PBS or PBS+Tween?
    I know they are many questions, but this is the first time I perform this kind of experiment.

    Thank you in advance,
    Best wishes

    Read More

    Abcam community

    Verified customer

    Asked on Feb 15 2017

    Answer

    Thank you for getting in touch.

    When using frozen sections, you can use a general frozen section staining protocol. I would amend the protocol booklet protocol as follows:

    1. Fix the sections (for example 4% PFA for 10 minutes or ice cold acetone for 5 minutes). Fixation will need to be individually optimized.

    2. Add enough drops of Hydrogen Peroxide Block to cover the sections. Incubate for 10 minutes. Wash 2 times in buffer.

    3.  Wash 3 times in buffer.

    4. Apply Protein Block (if required) and incubate for 10 minutes at room temperature to block nonspecific background staining. Wash 1 time in buffer.

    5. Apply primary antibody and incubate according to manufacturer’s protocol.

    6. Wash 4 times in buffer. Apply Biotinylated Goat Anti-Polyvalent and incubate for 10 minutes at room temperature. Wash 4 times in buffer.

    7. Apply Streptavidin Peroxidase and incubate for 10 minutes at room temperature.

    8. Rinse 4 times in buffer. Add 30 μl (1 drop) DAB Chromogen to1.5 ml (50 drops) of DAB Substrate, mix by swirling and apply to tissue. Incubate for 1-10 minutes. Rinse 4 times in buffer.

    9. Apply counterstain according to manufacturer’s instructions.

    10. Dehydrate if required and coverslip.

    We have further information on IHC protocols on the following web page which I hope you will find helpful:

    https://www.abcam.com/tag/ihc%20protocols

    Which primary antibody are you using? This kit can be used with rabbit primary antibodies. The antibody can be diluted in PBS 0.2% Tween and include the protein block,

    Wash buffer should be PBS 0.2% Tween

    I am happy to answer any further questions you have.

    Read More

    Abcam Scientific Support

    Answered on Feb 15 2017

    It is very easy to use, get sharp images with it.

    Excellent Excellent 5/5 (Ease of Use)
    Abreviews
    Abreviews
    abreview image
    This kit is very easy to use, just follow its instructions. It contains protein block, secondary antibody and substrate et al. in one box, basically everything you need for IHC except primary antibody. The box of the kit is of the right size, not too big not too small, so it is very easy to locate in the refrigerator. I wish the kit also contains Hematoxylin but it doesn't. The staining results are sharp and strong.

    Abcam user community

    Verified customer

    Submitted Nov 23 2016

    Question

    1) Why is the H2O2 treatment done before the antigen retrieval step?
    2) Why is the protein block only 5 min long?
    3) Why is the incubation with the 2nd antibody only 10 min long?

    Read More

    Abcam community

    Verified customer

    Asked on Dec 13 2012

    Answer


    The lab sent me the following explanations:

    The protocol for ab64261 reflects the sequence we follow in house. Personally I have done the endogenous peroxidase blocking step before antigen retrieval, after antigen retrieval and after the primary antibody and my sense is that it doesn’t matter when as long as it is before the HRP step. I do not have any experimental data to support that statement but that is my personal opinion.

    The Protein Block step can be used as necessary for up to 10 min to help decrease non-specific background. All incubation times were optimized during the development of this product.

    Read More

    Abcam Scientific Support

    Answered on Dec 13 2012

    Question

    I talked to you last week about my p-Met antibody (ab82094).  I recently bought a new vial and have an old vial I've been staining controls with, but have not been getting satisfactory results despite receiving the Dab Kit you gave me (AB64261).   I noticed that there was a protein block that is supposed to replace the BSA I used, but that didn't seem to help.  Do you suggest trying 5% BSA?  Before I was using 1% BSA.   Also, is there possibly an antigen retreival solution you recommend?  I just wanted to try all of your suggestions for reagants/controls because I can't seem to recreate what your labs have been able to get as far as results.  

    Read More

    Abcam community

    Verified customer

    Asked on Oct 26 2012

    Answer

    I am sorry that I was unavailable when you called but am glad to hear from you. We would not suggest using 5% BSA instead of the protein block included with the kit. These blocking solutions are formulated to give the best result with the kit. I feel that as you have continued to experience problem with the antibody that it may be that the vial or lot which you have received is faulty. I believe the lot which you have is xxx. We currently have a new lot available which I would be happy to send to you. Alternatively under our Abpromise guarantee I can credit/refund this product for you. https://www.abcam.com/index.html?pageconfig=abpromise I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

    Read More

    Abcam Scientific Support

    Answered on Oct 26 2012

    Question

    I recently purchased Rabbit specific HRP/DAB (ABC) detection IHC kit for CD31 (ab28364) primary antibody for mouse tissue section paraffin-embadded (50 micron paraffin section).
    I see that for this antibody, heat mediated antigen retrieval is recommended. Can you give me an instruction on this procedure? Will this step before or after peroxidase blocking?

    Also, i believe my sample slide is quite thick (50 micron) and i've tried to detect CD31 using invitrogen's AEC staining kit and unable to detect anything. So would you recommend to incubate primary antibody overnight or modulate any other steps of procedure?

    Also, would it be alright to use clear mount mounting solution from Invitrogen (#008010)?

    Read More

    Abcam community

    Verified customer

    Asked on Sep 28 2012

    Answer

    Thank you for contacting us. You will need an antigen retrieval step. Customers have reported success with a few different protocols but I recommend a heat mediated retrieval using a 10mM citrate buffer, pH 6.0. This should be applied to the sections before the peroxidase blocking step. After deparaffinization, simply heat the citrate buffer to boiling, immerse the re-hydrated slides in it, and maintain the buffer boiling, at 95-100C, for 20 minutes. Allow to cool for another 20 minutes and continue with the protocol. Here is a link to our IHC guide, which includes a section about antigen retrieval.

    https://www.abcam.com/index.html?pageconfig=resource&rid=11384

    The thickness of the sections may be an issue since the antibody recognizes an intracellular domain of CD31. If the diameters of the cells in the sections are less than 50 microns, then some will likely be intact, and the membranes of those cells needs to be permeabilized to allow the antibody access to the cytoplasmic domain of CD31. Include some detergent in the blocking buffer, for instance 0.1% Triton X-100, to permeabilize the cells.

    The Invitrogen mounting medium is aqueous, which is required for AEC. AEC dissolves in organic mounting media, resulting in no stain. The mounting medium is also compatible with the DAB in the ab24621 kit.

    I hope this helps. Please let me know if you have any questions.

    Read More

    Abcam Scientific Support

    Answered on Sep 28 2012

    Question

    xxxx;

    As I told you on the phone, I am planning on performing IHC on fresh PBMC samples from sheep.

    Cells will be isolated from whole blood using a density gradient and will be placed on glass slides using a cytocentrifuge. At this point, we CAN fix them if needed, but we don’t have to. What do you recommend?

    Our primary antibody is affinity purified rabbit polyclonal. We care considering using your EXPOSE Rabbit specific HRP-AES detection kit (ab94361) – do you think this is the best kit to use, or would you recommend a different one for our purposes? We are attempting to stain a transmembrane protein.

    We are planning to counterstain using hematoxylin: do you think we would get the best contrast with the AEC kit or would you recommend using DAB?

    Also, does abcam recommend purchasing hematoxylin from any particular vendor?

    If ab64230 the appropriate mounting medium to use with this kit?

    Thanks so much for your help. I look forward to your response!

    Read More

    Abcam community

    Verified customer

    Asked on Jun 20 2012

    Answer

    Thank you for replying with that information.

    As I mentioned over the phone, I have spoken to the source of these kits and we do not have any experimental data using either the ACE or DABdetection kits (ab64260, ab64261, ab80437 and ab94361)for IHC on fresh PBMC samples, asallof these kits have only been tested on formalin fixed paraffin embedded tissue samples.Wewould expect that as long as the primary antibody binds the target as expected that both kits could be suitable foryour samples but as we have not tested it ourselves we would not be able to guarentee this.



    There should be good contrast if using the AEC or DAB kit with hematoxylin as counterstain. We counterstain with hematoxylin in-house regardless ofwhether DAB or AEC is used. The sensitivity betweenAEC and DABis similar. I would recommend using ab94361 or ab80437 if you expect the PBMC samples to contain any endogenous biotin.I am sorry but I cannot recommend a particular supplier of hematoxylin.

    And to answer your final question, the mounting media ab64230 should be suitable for your purposes.

    I hope this information has been of help. If you require any further information please do not hesitate to contact us again.

    Read More

    Abcam Scientific Support

    Answered on Jun 20 2012

    1-10 of 16 Abreviews or Q&A

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