Avidin/Biotin Blocking Kit (ab64212)

Reviews (1)Q&A (3)References (20)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Avidin/Biotin Blocking Kit (ab64212)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Avidin/Biotin Blocking Kit (ab64212)
  • 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.

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 (Formalin/PFA-fixed paraffin-embedded sections) - Avidin/Biotin Blocking Kit (ab64212)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Avidin/Biotin Blocking Kit (ab64212)Image from Rodrigues et al., J Clin Invest., 128(10):4441-4453. doi: 10.1172/JCI121924. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/
    Micrographs showing T-cells infiltrationMulti-spectral, multicolor Immunofluorescence for T-cells infiltration in lymph nodes samples: dMMR tumor. (200x magnification). Multiplex sequential IF staining on 3-?m sections from FFPE tissue; Tissue sections were treated with an avidin/biotin blocking kit (ab64212) before using a biotin conjugated Foxp3 antibody. Other antibodies were conjugated to fluorochroms. CD4:red, CD8: green, CD4/Foxp3: tile, CD8 blue, EPCAM: yellow
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Avidin/Biotin Blocking Kit (ab64212)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Avidin/Biotin Blocking Kit (ab64212)Image Asano et al., PLoS One, 15(1):e0227814. doi: 10.1371/journal.pone.0227814. Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/
    Representative histological images of detours at the proximal surgical site. Sut: suture, V: vein, ColL: collecting LV, P: panniculus carnosus muscle, A: arterySpecimens for IHC staining of LVs were washed with PBS comprising 0.03% Tween-20 (PBST), incubated with protein blocking solution, endogenous avidin/biotin blocking kit (ab64212), and 0.03% hydrogen peroxide in methanol. The Syrian Hamster anti-podoplanin/gp36 antibody (ab11936) was used at a dilution of 1:600. Subsequently, the specimens were incubated with secondary antibodies linked with biotin (ab7145) followed by streptavidin linked with HRP (ab7403).
  • 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 (20)

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

ab64212 has been referenced in 20 publications.

  • Rescigno P  et al. Characterizing CDK12-Mutated Prostate Cancers. Clin Cancer Res 27:566-574 (2021). PubMed: 32988971
  • Coskun AF  et al. Nanoscopic subcellular imaging enabled by ion beam tomography. Nat Commun 12:789 (2021). PubMed: 33542220
  • Tsitrina AA  et al. Inhibition of Hyaluronan Secretion by Novel coumarin compounds and chitin synthesis inhibitors. Glycobiology N/A:N/A (2021). PubMed: 33978736
  • Lopez-Sanchez C  et al. Early Reactive A1 Astrocytes Induction by the Neurotoxin 3-Nitropropionic Acid in Rat Brain. Int J Mol Sci 21:N/A (2020). PubMed: 32443829
  • Asano K  et al. Pre-collecting lymphatic vessels form detours following obstruction of lymphatic flow and function as collecting lymphatic vessels. PLoS One 15:e0227814 (2020). PubMed: 31940420
View all Publications for this product

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!

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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.

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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.

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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.

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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.

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https://www.abcam.com/abreviews

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