I need a protocol for DAB staining in the cells. So, any method/ protocol suggested would be appreciated.
Thanks
Hi, I assume you want to perform some immunolocalization and to develop it with DAB, am I right?
As a general protocol you can try the following:
1- Wash with PBS 3 x 5 min
2- Endogenous peroxidase blocking 1% H2O2 in PBS 10 min
3- Wash with PBS 3 x 5 min.
4- Blocking with 10% BSA60 min @ 37 ̊C
5- Primary antibody in PBS-T/1% BSA 60 min @ 37 ̊C
6- Wash with PBS 3 x 5 min.
7- (peroxidase conjugated) Secondary antibody in PBS-T / 1% BSA 60 min @ 37 ̊C
8- Wash with PBS 3 x 5 min.
9- Incubate with DAB (under manufacturer's recommended conditions) until signal appears clearly developed. Be careful not to over-develop, otherwise background staining will become a problem. Include a positive and a negative control please.
10- Wash away DAB to stop reaction.
Please note DAB should be managed carefully as it is a suspected carcinogen. Handling and disposal must be according to Lunn and Sansone's protocol (Trends Genet. 1992 Jan;8(1):7).
Best of luck!
Most all protocols will be quite similar. I have attached the one I like using the best in our lab which calls for an overnight incubation of primary antibody. It also includes a counterstain. The unmasking solution to which it refers is from Vector Labs (cat# H-3300). The DAB that is used in the protocol is from Dako (cat# K3468). It is a liquid, but if you have one of the other versions, just mix it up according to manufacturer's instructions.
I recommend this patent, that scrutinized DAB staining conditions. https://patents.google.com/patent/WO2010077870A2/en11 Stable compositions comprising chromogenic compounds and methods of use
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