Immunohistochemical Staining Method for Phospho-proteins in Paraffin Sections

Deparaffinization and re-hydration of tissue slide

Before deparaffinization, place the slides in a 55°C oven for ten minutes to melt the paraffin.
Deparaffinize slides in 2 changes of xylene or xylene substitute for 5 min each.
Transfer slides to 100% alcohol, 2 changes for 3 minutes each and transfer once through 95% alcohol for 3 min.
Block endogenous peroxidase activity by incubating sections in 3% H 2O 2 solution in methanol for 10 min.
Rinse in PBS 2X for 5 min each time.
If the antibody staining requires antigen retrieval to unmask the antigenic epitope refer below to section 2. If antigen retrieval is not required proceed to section 3.

Pretreatment of paraffin sections with BD Retrievagen A (pH 6.0) (cat. no. 550524):

Make a working solution of Retrievagen A by mixing 18 ml of Retrievagen A solution 1 and 82 ml of Retrievagen A solution 2 and bring the final volume to 1 liter in distilled water.
Place slides in a plastic coplin jar filled with the working Retrievagen solution and heat in a microwave oven to 203°F (95°C) (microwave oven or other heating sources such as pressure cooker (see alternate protocol), water bath can be used).
Mix the working Retrievagen A solution in the coplin jar with a disposable pipet and incubate the slides at 203°F for 10 min.
Remove the coplin jar with the slides, cover the jar tightly, and allow the solution to slowly cool to room temperature for 20 min.
Note: It is important to let the temperature ramp down slowly to allow the protein molecules to fold properly.
Rinse slides in PBS 3X, 5 minutes each time.

Alternate protocol for antigen retrieval

For antigen retrieval using a pressure cooker or an autoclave, pretreat slides with BD Retrievagen A solution in a glass or metal coplin jar as outlined in step 2 above.
Heat coplin jar containing slides with BD Retrievagen A solution in a pressure cooker or autoclave at 120-125°C, 17-25 psi for 5 minutes.
When completed (at 0 psi), open pressure cooker or autoclave and allow slides to cool to room temperature (approximately 20-30 min) prior to removing them from the coplin jar.
Wash slides as indicated in step 2 above.
Block with avidin for 15 min.
Wash with PBS twice for 3 min each.
Block with biotin for 15 min.
Wash with PBS 3 times for 3 min each.
Treat slide (if needed) with CIP or Lambda phosphatase (50 µg/ml at 37°C, 45 min).
Wash the treated slides with Tris buffer 3 times.
Wash with PBS once 3 min.
Incubate with primary antibody for 1 hr at RT or (over night at 4°C).
Wash with PBS 3 times for 3 min each.
Incubate with secondary antibody for 30 min.
Wash with PBS 3 times for 3 min each.
Incubate with streptavidin-HRP for 30 min.
Wash with PBS 3 times for 3 min each.
Development with DAB solution (less than 5 min).
Dewater to clean (about 30 min).
Dehydrate through 4 changes of alcohol (95%, 95%, 100% and 100%).
Clear in 3 changes of xylene (or xylene substitute).
Counter stain with hematoxylin.
Add coverslip.

Injection Method

Prepare 5 mM sodium vanadate in PBS by heating to boiling.
Fifteen minutes prior to use, add 30% H 2O 2 to the vanadate solution at room temperature to a final concentration of 50 mM.
Inject solution of peroxovanadate or PBS intraperitoneally into adult rat at a dose of 10 µl/g of body weight.
Sacrifice rats, remove tissues, and immediately put into formalin fixative, zinc fixative, or frozen blocks. For detailed protocols on staining of zinc fixed or frozen sections, please refer to Immunohistochemistry section of our protocols at: bdbiosciences.com/resources/protocols

Additional information and buffers used in the protocol:

For avidin and biotin blocking, we recommend VECTOR Laboratories Kit (Avidin/Biotin Blocking Kit, Sp-2001).

Avidin and biotin blocking is recommended for tumor sections that contain a lot endogenous biotin.

Tris Buffer:

Tris-HCl, 1.0 M (pH 8.0) (Sigma T-3038): 10.0 ml
Magnesium chloride (MgCl2), 100 mM: 10.0 ml
Zinc chloride (ZnCl2), 10 mM: 1 ml
Sodium chloride (NaCl), 5 M: 30 ml
DdH 2O: to 1000 ml (1L)