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    products/chromogen-kits/dab-substrate-kit-ab64238.pdf

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DAB Substrate Kit (ab64238)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (7)Q&A (18)References (195)

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)

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Overview

  • Product name

    DAB Substrate Kit
  • Product overview

    DAB substrate kit ab64238 is used for IHC staining using HRP / peroxidase enzyme for detection. Reagent is stable for up to 6 hours after mixing the two components.


    DAB (3,3'Diaminobenzidine) is the most commonly used chromogen for immunohistochemical staining. In the presence of HRP / peroxidase, DAB produces a brown precipitate that is insoluble in alcohol.


    This product is a two component form consisting of a liquid, refrigerator stable DAB Chromogen and DAB Substrate.  


    IHC protocol suitable for use with DAB substrate kit:
    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, 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 and incubate for 10 minutes at room temperature.


    8. Rinse 4 times in buffer. Place slide in DAB substrate ab64238 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.

  • Notes

    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-Pmore details

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Storage buffer

    Constituents: 100% Propylene glycol, 5% Hydrogen peroxide, DAB substrate buffer
  • Components 60 ml 125 ml
    50x DAB Chromogen 1 x 2ml 1 x 4ml
    DAB substrate 1 x 60ml 1 x 125ml
  • Research areas

    • Kits/ Lysates/ Other
    • Tools and Reagents
    • IHC Tools/ Reagents
    • Kits/ Lysates/ Other
    • Kits
    • IHC tools
    • DAB Substrate
  • Relevance

    3,3' Diaminobenzidine (DAB) is a widely used chromogen for immunohistochemical staining. In the presence of peroxidase enzyme, DAB produces a brown precipitate that is insoluble in alcohol.
  • Alternative names

    • 3 3' Diaminobenzidine

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab64238 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 dilution.
Notes
IHC-P
Use at an assay dependent dilution.

Images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)Lean QY et al. PLoS One (2015) Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    Immunohistochemical analysis of healthy and colitis mouse colon sections (untreated and treated with enoxaparin) labeling claudin-4 with ab15104 at 1/200. ab7090, anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (HRP) at 1/300 was used as the secondary antibody. ab64238 at 1/50 was used to develop the histological signal.

    Antigen retrieval was performed by incubating the sections for 10 minutes at 97°C in 1 mM EDTA buffer, pH 8.0 or 10 mM citrate buffer, pH 6.0.

    Control, C; untreated colitis, DSS; oral enoxaparin, OE; intraperitoneal injection of enoxaparin, IPE.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)Kumar P et al. PLoS One (2014) Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    Immuno-staining of formalin–fixed and paraffin-embedded liver sections with ab64238. Sections were obtained from control (saline) and treated with CCl4+CT-N and CCl4+AD-N (left) and antibody control (right). 

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)Jujo T et al. PLoS One (2014) Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    Immunohistochemical analysis of mouse lung. Samples were fixed in 10% buffered formalin, paraffinized and sliced at 1.5 µm thick. Antigen retrieval was performed using ab64214 for the deparaffinized slices. Sections were blocked with 2% normal goat serum, PBS(-) and 0.1% Tween20. They were then incubated with the primary antibodies for 1 hour at 4°C and with secondary antibodies for 30 minutes at room temperature. The avidin-biotin-peroxidase complex method with peroxidase streptavidin and the DAB substrate kit ab64238 was performed.

    (A) A resected lung from a mouse sacrificed 28 days after SCL injection.

    (B) Haemotoxylin and Eosin staining showing the tumor composed of a central area with necrosis and a peripheral zone filled with SCLs.

    (C) Immunohistochemical staining for MMP-14 showing positive expression of MMP-14 in the peripheral zone of the tumor and negative in central zone.

    (D) Immunohistochemical staining weak staining of MMP-2 in the peripheral zone. 

Protocols

  • DAB Substrate Kit
  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (195)

Publishing research using ab64238? Please let us know so that we can cite the reference in this datasheet.

ab64238 has been referenced in 195 publications.

  • Liu J  et al. Cancer-Associated Fibroblasts Provide a Stromal Niche for Liver Cancer Organoids That Confers Trophic Effects and Therapy Resistance. Cell Mol Gastroenterol Hepatol 11:407-431 (2021). PubMed: 32932015
  • Dolicka D  et al. Tristetraprolin Promotes Hepatic Inflammation and Tumor Initiation but Restrains Cancer Progression to Malignancy. Cell Mol Gastroenterol Hepatol 11:597-621 (2021). PubMed: 32987153
  • Kopaeva MY  et al. Pretreatment with Human Lactoferrin Had a Positive Effect on the Dynamics of Mouse Nigrostriatal System Recovery after Acute MPTP Exposure. Biology (Basel) 10:N/A (2021). PubMed: 33401480
  • Han C  et al. Berbamine Suppresses the Progression of Bladder Cancer by Modulating the ROS/NF-?B Axis. Oxid Med Cell Longev 2021:8851763 (2021). PubMed: 33520087
  • Zuo S  et al. Enhanced antitumor efficacy of a novel oncolytic vaccinia virus encoding a fully monoclonal antibody against T-cell immunoglobulin and ITIM domain (TIGIT). EBioMedicine 64:103240 (2021). PubMed: 33581644
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 25 Abreviews or Q&A

Visualization step using ab64238 DAB substrate.

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
Ki67 IHC staining on rat bladder, and visualization step using ab64238 DAB substrate (1:50 dilution, 1 min incubation time). We are satisfied with the results.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted May 27 2022

DAB with anti PLP

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
Gives good results at a dilution 1:50. Used on brain sections with an antibody against PLP
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

MISS. Emily Santrau

Verified customer

Submitted Sep 23 2020

DAB substrate for IHC staining

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
CNPase (ab6319) staining on murine brain tissue. Visualization step using ab64238 DAB substrate (1:50 dilution, 10min incubation time)
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Jul 29 2020

DAB substrate for IHC

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
Excellent DAB substrate
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted May 29 2020

A review of KLF4 staining of prostate cancer

Good Good 4/5 (Ease of Use)
Abreviews
Abreviews
abreview image
This DAB substrate kit (ab94665) is easy to use, and the quality is good.


Liyong Zhang
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Aug 15 2017

Abreview for DAB substrate kit

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
This kit is streamlined to use at ease. The results is fantastic.


Liyong Zhang
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Aug 15 2017

IHC-P Mouse pancreas

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
Ki67 IHC staining on mouse pancreas, blocking with 10% BSA, 1 hr ab incubation RT
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Aug 16 2016

Question

Thank you for your prompt response. Do you recommend any buffer to go with the heat mediated retrieval, citrate, Tris/EDTA? DAB detection system would suit, I see you have detection kits such as https://www.abcam.com/Mouse-and-Rabbit-Specific-HRPDAB-ABC-detection-IHC-kit-ab64264.html that include secondary antibody.

I will performing immunohistochemisty using DSG3 (3G133), p63 (4A4) and RTNK2 (2C2) and if possible would like a detection kit to suit all. Would https://www.abcam.com/Mouse-and-Rabbit-Specific-HRPDAB-ABC-detection-IHC-kit-ab64264.html be suitable for this?

Read More

Abcam community

Verified customer

Asked on Dec 06 2012

Answer

Thank you for contacting us.
The kit will work with the rabbit and mouse primary antibodies.
You can try citrate buffer pH for a start and Tris/EDTA in case the staining does not work. Please check the following for protocol and troubleshooting of;
https://www.abcam.com/index.html?pageconfig=resource&rid=11384
https://www.abcam.com/index.html?pageconfig=resource&rid=11467
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Read More

Abcam Scientific Support

Answered on Dec 06 2012

Question

I am enquiring about Rhotekin 2 primary antibody for use with IHC-P. I see you have an antibody clone 2C2, can you provide any information on appropriate epitope retrieval methods and detection kits or secondary antibody that would be suitable for use with this antibody?

Read More

Abcam community

Verified customer

Asked on Dec 05 2012

Answer

Thank you for contacting us.
Heat mediated antigen retrieval method with microwave or pressure cooker is recommended for this ab.
The suitable detection system depends on the substrate you want to use. e.g. with DAB or AEC, we have following products;
https://www.abcam.com/DAB-Enhancer-ab675.html
https://www.abcam.com/DAB-Substrate-Kit-ab64238.html
https://www.abcam.com/AEC-Substrate-System-Ready-to-Use-ab64252.html
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Read More

Abcam Scientific Support

Answered on Dec 05 2012

Question

It is not idea of searching the items.. I told you that we need all reagents needed to do full IHC experiment based on your technical experience.. So if we need more items other than the ones who sent to me, please let me know.. Like antibosy diluent, antigen retrival .. etc, The customers is new to IHC and we are not experts in IHC as well.. So please advice..



It is ok about the monoclonal rabies, waiting your kind e-mail !



Truly yours,

Omar

Read More

Abcam community

Verified customer

Asked on Aug 03 2012

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/Rabies-Virus-antibody-1C5-ab1002.html (or use the following: https://www.abcam.com/Rabies-Virus-antibody-1C5-ab1002.html).


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


https://www.abcam.com/Goat-Mouse-IgG2a-heavy-chain-Biotin-preadsorbed-ab98696.html (or use the following: https://www.abcam.com/Goat-Mouse-IgG2a-heavy-chain-Biotin-preadsorbed-ab98696.html).


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.

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

Read More

Abcam Scientific Support

Answered on Aug 03 2012

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