DAB Substrate Kit (ab64238)
Overview
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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.
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Notes
Find complete IHC kits, and reagents for antigen retrieval, blocking, signal amplification, visualization, counterstaining, and mounting in the IHC kits and reagents guide.
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Tested applications
Suitable for: IHC-Pmore details
Properties
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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
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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
Images
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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.
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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. Secontions were obtained from control (saline) and treated with CCl4+CT-N and CCl4+AD-N (left) and antibody control (right).
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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.