DAB Substrate Kit (ab64238)
Product nameDAB Substrate Kit
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.
Find complete IHC kits, and reagents for antigen retrieval, blocking, signal amplification, visualization, counterstaining, and mounting in the IHC kits and reagents guide.
Tested applicationsSuitable for: IHC-Pmore details
Storage instructionsStore at +4°C. Please refer to protocols.
Storage bufferConstituents: 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
Relevance3,3' 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.
- 3 3' Diaminobenzidine
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.
Use at an assay dependent dilution.
Use at an assay dependent dilution.
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.
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).
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.
Datasheets and documents
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