Avidin/Biotin Blocking Kit (ab64212)

Reviews (1)Q&A (3)References (6)
Immunohistochemistry - ab64212

    Overview

    • Product name

      Avidin/Biotin Blocking Kit

    • Product overview

      Avidin Biotin Blocking Kit ab64212 blocks signal from endogenous avidin, biotin and biotin-binding proteins in tissues when used with biotin-based IHC detection (eg. with ABC IHC detection kits).


      When using the kit, firstly an excess of avidin is added to the sample to bind endogenous biotin, that avidin is then blocked with an excess of biotin. Excess biotin and avidin is washed away.


      The kit is often used with cells and tissues containing high levels of biotin. This can be indicated by blocking sections with hydrogen peroxide, and then incubating sections with streptavidin-HRP and then DAB; brown DAB staining indicates endogenous biotin. Kidney, liver, spleen especially contain high levels of biotin.


      This kit was previously called Endogenous Avidin/Biotin Blocking Kit.



      IHC protocol suitable for use with Avidin Biotin Blocking Kit ab64212:
      For frozen sections, skip steps 1 and 2. 


      1. Deparaffinize and rehydrate formalin-fixed paraffin-embedded tissue section.


      2. Use appropriate antigen retrieval buffer or enzyme (primary antibody dependent) to treat sections. Wash 3 times in buffer.


      3. Add enough hydrogen peroxide blocking solution to cover the sections. Incubate for 10 minutes. Wash 2 times in buffer. If necessary, block for endogenous biotin by incubating with avidin block for 15 mins, washing twice, incubating with biotin block for 15 mins, and washing twice.


      4. Apply protein block (or normal serum from same species as secondary antibody) and incubate for 5 minutes at room temperature to block nonspecific background staining. Wash once in buffer.


      5. Apply primary antibody in antibody diluent and incubate.


      6. Wash 4 times in buffer. Incubate slide with biotinylated secondary antibody (or HRP polymer secondary antibody and skip step 7). Wash 4 times in buffer.


      7. Apply streptavidin-HRP and incubate for 10 minutes at room temperature.


      8. Rinse 4 times in buffer. Place slide in DAB substrate or AEC Substrate and incubate until desired color is achieved (1-10 mins). Rinse 4 times in buffer.


      9. Add enough drops of hematoxylin to cover the section. Incubate for 1 minute.


      10. Rinse 7-8 times in tap water. Add mounting medium to cover the section.



      Find complete IHC 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, ICC/IF, IHC-Frmore details

    Properties

    • Storage instructions

      Store at +4°C. Please refer to protocols.

    • Storage buffer

      Preservative: 0.08% Sodium azide
      Constituent: Avidin
    • Components 15 ml
      Avidin Block 1 x 15ml
      Biotin Block 1 x 15ml

    • Research areas

    • Relevance

      Some cells, and tissues such as kidney, liver and spleen, contain endogenous biotin. Using an avidin-biotin staining method may result in high, non-specific background staining. A significant reduction of this non-specific background can be obtained by pre-treatment of cells/tissues with avidin/biotin blocking reagents prior to the incubation of biotinylated antibody.

    Associated products

      Applications

      The Abpromise guarantee

      Our Abpromise guarantee covers the use of ab64212 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.
      ICC/IF
      Use at an assay dependent concentration.
      IHC-Fr
      Use at an assay dependent concentration.
      Notes
      IHC-P
      Use at an assay dependent concentration.
      ICC/IF
      Use at an assay dependent concentration.
      IHC-Fr
      Use at an assay dependent concentration.

      Images

      • Immunohistochemistry - ab64212
        Immunohistochemistry - ab64212Image from Filliat G., PLoS One 12(7). Fig 6a & b. doi: 10.1371/journal.pone.0181600. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

        Immunohistochemical analysis staining HTRA1 in mouse bone tissue sections. Tissue sections were dewaxed, rehydrated and treated with Endogenous Avidin/Biotin Blocking Kit (ab64212), 3% H2O2 and normal swine serum. Tissue sections were then incubated with a polyclonal anti-HTRA1 antibody for 1 hour at 37°C. After PBS wash, tissue sections are incubated with biotinylated swine anti-rabbit IgG for 45 minutes at 37°C and further incubated for 30 minutes after washing. With Vectastatin. Sections were developed using DAB and counterstained using Harris modified hematoxylin. 

      References (6)

      Publishing research using ab64212? Please let us know so that we can cite the reference in this datasheet.

      ab64212 has been referenced in 6 publications.

      • Peng C  et al. Synchronous primary sigmoid colon cancer and primary thyroid cancer followed by a malignant tumor of the kidney: Case report of multiple primary cancer and review of the literature. Oncol Lett 17:2479-2484 (2019). PubMed: 30719116
      • Teissier T  et al. Knockout of receptor for advanced glycation end-products attenuates age-related renal lesions. Aging Cell 18:e12850 (2019). PubMed: 30794349
      • Yang H  et al. Predictive and prognostic value of phosphorylated c-KIT and PDGFRA in advanced non-small cell lung cancer harboring ALK fusion. Oncol Lett 17:3071-3076 (2019). PubMed: 30867736
      • Hammad S  et al. Confounding influence of tamoxifen in mouse models of Cre recombinase-induced gene activity or modulation. Arch Toxicol 92:2549-2561 (2018). PubMed: 29974145
      • Filliat G  et al. Role of HTRA1 in bone formation and regeneration: In vitro and in vivo evaluation. PLoS One 12:e0181600 (2017). IHC . PubMed: 28732055
      • Planque SA  et al. Physiological IgM class catalytic antibodies selective for transthyretin amyloid. J Biol Chem 289:13243-58 (2014). PubMed: 24648510

      Customer reviews and Q&As

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      1-4 of 4 Abreviews or Q&A

      helpful kit

       Excellent Excellent 5/5 (Ease of Use)
      Abreviews
      We used this kit to block the non-specific binding of avidin or biotinylated reagents with the endogenous biotin, also their receptors and the avidin binding sites in the tissue.
      Its works good and is easy to use.
      We used the kit as a ready to use-kit and incubated the samples first with the avidin block for 10minutes and after a washing step with the bioin block for 10minutes.

      Abcam user community

      Verified customer

      Submitted Mar 12 2018

      Question


      I just purchased the avidin/biotin blocking kit
      and I have a question regarding the protocol for this product:
      I do immunohistochemistry on free floating sections,
      typically with my sections floating in 3mL of PBS with 1%BSA.
      How many drops of the avidin and biotin solution do you
      recommend adding to my incubations?
      Is 3 drops of blocking solution
      diluted in 3mL PBS enough for successful blocking?
      I would really appreciate your help!

      Read More
      Answer


      I can confirm that the Avidin Block and Biotin block provided with this kit come ready to use. They should not be diluted.

      Once the sections are cut into sections and placed on a slide, the avidin and biotin blocking is done as part of the antibody staining procedure. You will need to pipette enough solution on to cover the tissue section and ensure it does not dry out during incubation. Typically this is usually 50 - 200 ul depending on the size of the section.

      There is a protocol provided on the datasheet, see the link in the protocols tab.

      Incubate tissue sections with Avidin Block for 10-15 minutes.
      Wash 2 times with buffer.
      Incubate tissue sections with Biotin Block for 10-15 minutes.
      Wash 2 times with buffer
      Apply Primary antibody to the tissue sections


      I hope this will be helpful. If you have any further questions, please do not hesitate to contact me.

      Read More

      Question

      I have one more questions. I am not sure if I need to use both normal serum and biotin blocking kit. Because the primary antibodies bind non specifically to random proteins as well as the streptavidin probe binds to the endogenous biotin.

      Read More
      Answer

      Thank you for contacting us.

      I would not recommend using the normal serum after biotin blocking for fear of introducing biotin to the samples. However if you would like, you may block with normal serum (5-10%) prior to biotin/avidin blocking. As you are not using a secondary you can use any normal serum at hand, donkey, goat, etc.

      I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

      Use our products? Submit an Abreview. Earn rewards!
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      Read More

      Question

      Thank you for your reply (below) to my student, regarding serum for use in IHC staining. I am the PI on the project the student is working on, and I thought I'd give some details on what we're doing so as to clarify things. We are interested in imaging FFPE tissue sections using a combination of luminescent probes and a novel microscopy system. For proof-of-principle experiments, we purchased some FFPE control slides and corresponding biotin-conjugated antibodies (see list below). Our luminescent probe is conjugated to streptavidin. So, what we we're hoping to do is: 1) Prepare FFPE section using variation of IHC-P protocol. 2) Stain with primary antibody (biotin-conjugated) 3) Stain with probe-conjugated streptavidin as secondary 4) Image. From these experiments we hope to see what sorts of Signal-to-background ratio, signal-to-noise ratio we get in images taken with our probe vs. control fluorescence microscopy images obtained using fluorescein-conjugated streptavidin. I guess what we need to know is what sorts of blocking agents and/or serum we should use. Obviously, we want to have as little non-specific binding of either primary antibody or streptavidin to tissue.

      Read More
      Answer

      Thank you for your reply.

      In this case I would recommend that you quench endogenous biotin/avidin in your tissue prior to staining. We recommend use of our Endogenous Avidin/Biotin Blocking Kit (ab64212).

      After blocking with this system, you may incubate your primary antibodies diluted in PSB or TBS.

      Use of a mild detergent in your wash solution may also help to reduce background but isn't always neccessary.

      More information about our Endogenous Avidin/Biotin Blocking Kit (ab64212) may be found at the location below:

      https://www.abcam.com/Endogenous-Avidin-Biotin-Blocking-Kit-ab64212.html


      I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

      Use our products? Submit an Abreview. Earn rewards!
      https://www.abcam.com/abreviews

      Read More

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