Overview

  • Product name

  • 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.

Applications

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.

Images

  • Immunohistochemical analysis staining HTRA1 in mouse bone tissue sections. Tissue sections were dewaxed, rehydrated and treated with Endogenous Avidin/Biotin Blocking Kit (ab64212), 3% H2Oand 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

This product has been referenced in:

  • 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). Read more (PubMed: 30719116) »
  • Teissier T  et al. Knockout of receptor for advanced glycation end-products attenuates age-related renal lesions. Aging Cell 18:e12850 (2019). Read more (PubMed: 30794349) »
See all 6 Publications for this product

Customer reviews and Q&As

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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

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