Overview

  • Product name

    Streptavidin HRP (ready-to-use)
  • Tested applications

    Suitable for: IHC-Pmore details
  • General notes

    Streptavidin-HRP for use with biotinylated secondary antibodies during IHC / immunohistochemistry. Optimized, ready-to-use format.

    This product was previously called Streptavidin-Peroxidase.

     

    IHC protocol suitable for use with Streptavidin-HRP ab64269:

    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, use avidin biotin blocking.

    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 ab64269 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. 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.

Properties

Applications

Our Abpromise guarantee covers the use of ab64269 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.

Images

  • ab64269 Straptavidin Peroxidase used along with an anti-CD20 antibody on Human Tonsil
  • Human colon carcinoma stained with anti-p53 antibody using Streptavidin Peroxidase (ab64269).

References

This product has been referenced in:

  • Petrovic R  et al. Mouse strain and sex as determinants of immune response to trivalent influenza vaccine. Life Sci N/A:N/A (2018). Read more (PubMed: 29859986) »
  • Pokharel A  et al. Asymmetric development of the male mouse mammary gland and its response to a prenatal or postnatal estrogen challenge. Reprod Toxicol 82:63-71 (2018). Read more (PubMed: 30315872) »
See all 6 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Abreviews
paraffin-embedded mouse/rat mandible/tibia/femur IHC staining에 사용

Miss. myeonga kim

Verified customer

Submitted Jan 24 2018

Answer

Thank you for contacting us again.



ab1002 Anti-Rabies Virus antibody looks very promising as it has been established for the use in IHC-P and should detect a wide range of rabies strains. As a starting dilution the customer can try a 5 ug/ml dilution incubated overnight at 4C. However, it may be that the actual optimum dilution may be lower or higher, but this depends on her samples. Another point is that I would strongly recommend a heat induced Antigen Retrieval if her slides are fixed with formalin.


https://www.abcam.com/index.html?datasheet=1002 (or use the following: https://www.abcam.com/index.html?datasheet=1002).


A matching secondary antibody would be Goat polyclonal Secondary Antibody to Mouse IgG2a - heavy chain (Biotin), pre-adsorbed (ab98696)


https://www.abcam.com/index.html?datasheet=98696 (or use the following: https://www.abcam.com/index.html?datasheet=98696).


To be fair, I am not the biggest fan of ready- to- use solution (andmost of my colleagues here a neither), for the following reason: most of all antibodies need optimization, may it be the blocking step, the retrieval time or method, the antibody incubation buffer etc. With ready-mix solutions it almost impossible to adjust the experiment settings. Ready mix solution make sense if the antibody is used in a high -throughput lab (like 20 slides at once with a fixed protocol), but not if you have to "learn" IHC-P and have the freedom to adjust the protocol fore the perfect results. In addition, it's cheaper ;)


Of course, once the protocol is optimized, using ready mixed solution are a very time efficient and make life easier, but I wouldn't use them as a beginner.


All the buffers your customer needs (see attachment) are listed in our very detailed and easy to follow step-by-step our protocol. However, none of my advice is written in stone, so if your customer prefers the ready mix solutions, we are more than happy to sell them to her.


Something we cannot provide are the reagents for the de-paraffinization, I am afraid, but a company like Sigma should be able to help with that.


Pease sent the customer our protocol and my advice. I am more than happy to assist her to get her experiments running. And another thing: she shouldn't be shy to ask any question she may have about the protocol!

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

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Answer

Thank you for contacting us.

Here is the list with the requested items. However, please be aware that this is not a technical inquiry as all item could be found by using the search application on our website under the "reagents" category. Please let us know if you need help on navigating our website so you can help your customers faster than relying on us.


Regarding the Rabies antibody, I am afraid we do not have what your customer requests: All we have are mouse monoclonal antibodies against this virus, but none produced in goat. Please let me know if this expectable for her, so I can suggest the right primary antibody with the matching biotinylated secondary antibody.


https://www.abcam.com/index.html?datasheet=64214 (or use the following: https://www.abcam.com/index.html?datasheet=64214).

https://www.abcam.com/index.html?datasheet=64216 (or use the following: https://www.abcam.com/index.html?datasheet=64216).

https://www.abcam.com/index.html?datasheet=64238 (or use the following: https://www.abcam.com/index.html?datasheet=64238).

https://www.abcam.com/index.html?datasheet=64269 (or use the following: https://www.abcam.com/index.html?datasheet=64269).

https://www.abcam.com/index.html?datasheet=94666 (or use the following: https://www.abcam.com/index.html?datasheet=94666).

https://www.abcam.com/index.html?datasheet=128988 (or use the following: https://www.abcam.com/index.html?datasheet=128988).

https://www.abcam.com/index.html?datasheet=128990 (or use the following: https://www.abcam.com/index.html?datasheet=128990).



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

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Answer

Thank you for contacting us.

We do not currently offer double staining IHC kits. Your staining conditions are challenging as, of the two common HRP chromogens, only DAB is alcohol insoluable. While of the common AP chromagens, only BCIP/NBT is insoluable. This means that to do double labeling you will have to do a sequential stain first using one detection system and then with the other.

I have collected a list, with links, of the reagents which you would need for this type of staining.

1) After de-parrafinizing and re-hydrating the samples, perform an antigen retrieval step treating with Cirate buffer pH6.0,https://www.abcam.com/10x-Citrate-Buffer-pH-6-0-ab64214.html, in the microwave or pressure cooker. After performing the antigen retrieval rinse in cold tap water for 10 minutes.

2) Rinse slides in TBST,https://www.abcam.com/20x-TBS-T-with-Tween-20-ab64250.html.

3) Performing a blocking step using protein block,https://www.abcam.com/Protein-Block-ab64226.html.

4) Incubate sections with your first primary antibody, for clarity I will assume the CD44 antibody, raised in mouse.

5) Wash with TBST.

6) Block endogenous peroxidases using aHydrogen Peroxide Blocking Reagent,https://www.abcam.com/Hydrogen-Peroxide-Blocking-Reagent-ab94666.html.

7) Incubate your tissue with a biotinylated secondary antibody, in this instance anti-Mouse IgG,https://www.abcam.com/Biotinylated-Goat-anti-Mouse-IgG-H-L-Ready-to-Use-ab64255.html.

8) Wash with TBST.

9) Detect your first antibody usingStreptavidin Peroxidase (Ready to Use)https://www.abcam.com/Streptavidin-Peroxidase-Ready-to-Use-ab64269.html. Note that this does not containa serum or BSA solution as diluent. Serum may contain biotin therefore competing Streptavidin binding with biotinylated secondary antibody, therefore reducing binding activity.

10) Apply your chromogen, DABsubtrate,https://www.abcam.com/DAB-Substrate-Kit-ab64238.html.

11) Wash with TBST.

12)Performing a second blocking step using protein block,https://www.abcam.com/Protein-Block-ab64226.html.

13)Wash with TBST.

14)Incubate sections with the your second primary antibody, this time ALDH1, raised in rabbit.

15)Wash with TBST.

16)Incubate your tissue with a biotinylated secondary antibody, in this instance anti-Rabbit IgG,https://www.abcam.com/Biotinylated-Goat-Anti-Rabbit-IgG-H-L-Ready-to-use-ab64256.html

17)Wash with TBST.

18)Detect your first antibody usingStreptavidin Alkaline Phosphatase (Ready to Use)https://www.abcam.com/Streptavidin-Alkaline-Phosphatase-Ready-to-Use-ab64268.html.

19) Apply your second chromogenAlkaline Phosphatase chromogen (BCIP/NBT) - Ready to Use,https://www.abcam.com/Alkaline-Phosphatase-chromogen-BCIP-NBT-Ready-to-Use-ab7468.html.

18) Wash with TBST, counterstain if desired.


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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Answer

Merci de nous avoir contactés.

Les deux kits que je vous recommende sont :

Streptavidin Alkaline Phosphatase (Ready to Use), www.abcam.com/ab64268
Streptavidin Peroxidase (Ready to Use), www.abcam.com/ab64269



Je vous fais parvenir un devis dans un autre email.

J'espère que ces informations vous seront utiles. N'hésitez pas à nous contacter de nouveau si vous avez d'autres questions.

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