1.        Grow cells on sterile coverglasses or chamber slides overnight.

2.        Rinse briefly with PBS

3.       Fix cells by incubation with one of the following methods:

4.       Rinse three times with PBS

5.        Incubate with 1% H₂O₂ in PBS for 10 minutes to quench endogenous peroxidase activity

6.        Rinse with PBS twice

7.       Incubate slides for 30 minutes in 10% blocking serum in PBS (suppresses non-specific binding of IgG)

8.       Blot excess blocking serum

9.        Incubate with primary antibody for 1 hour at room temperature or overnight at 4¡ãC. Optimal antibody concentration is usually from 0.5-10ug/ml in TBS.

10.    Wash three times with PBS

11.    Incubate with biotin-conjugated secondary antibody for 30-45 minutes at room temperature. Again, optimal antibody concentration is usually from 0.5-10ug/ml in TBS

12.    Wash three times with PBS

13.    Incubate with streptavadin-HRP for 15 minutes. This step should be titrated as excess streptavadin-HRP can lead to high background

14.   Wash with PBS three times

15.    Incubate with freshly mixed DAB solution for 3-10 minutes. We highly suggest that you should observe the color developing under microscopy.

16.    Rinse three times with distilled water

17.   Counterstain with hematoxylin

18.    Dehydrate with 70-100% alcohol and clear with xylene

19.    Mount with coverslip

Other Protocols:

A. DakoCytomation Envision Doublestain System.  

http://www.dakousa.com/prod_downloadpackageinsert.pdf?objectid=105439003

B. Handbook of Immunochemical Staining Methods.(3rd edition,DAKO)

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