RE: Keratin rapid IHC testing

From:Tony Henwood

RE: Keratin rapid IHC testing

Below is our protocol. Use the antibody at your usual dilution. Use ethanol fixation. No enzyme or HIER retrieval.

Tony Henwood JP, BappSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at  Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: (02) 9845 3306
Fax: (02) 9845 3318

-----Original Message-----
From: Poteete, Jacquie A. []
Sent: Wednesday, 5 March 2003 8:45
To: ''
Subject: Keratin rapid IHC testing

Hi everyone,

We are beginning to get inquiries for IHC rapid testing (15-20 minutes) of
frozen tissue and touch preps for Keratin.  Am I the only person who doesn't
know how to do this?  Does anyone have a protocol they would be willing to
share?  Any good references? Thanks for any help you might provide.

Jacquie Poteete MT(ASCP), IHC Lead Technologist
Saint Francis Hospital, Tulsa, OK

Document Procedure:            
Rapid immunoperoxidase Method for frozen sections

Often, it is required to perform a rapid IPX reaction on frozen sections in order to clarify a diagnosis. The following works well and allows results in 20-30 minutes.

Antigens differ in their susceptibility to fixation. Current studies in the department indicate that some antigens are best preserved with ethanol, whereas others require air-drying followed by cold acetone treatment.

Ethanol Fixation
Caution Flammable

1. Cut frozen sections at 5um thickness.
2. Place sections on Sticky slides
3. Immediately fix slides in 95% ethanol for 20 minutes.
4. Place sections in Endogenous Block solution and continue with the procedure as described below.

Air Dried Procedure
Caution Flammable

1. Place a coplin jar containing 20ml acetone in the -20oC Freezer
2. Cut frozen sections at 5um thickness.
3. Place sections on Sticky slides
4. Air dry sections for 15minutes
5. Place sections in cold acetone and allow to warm up to room temperature (10minutes).
6. Remove sections from acetone and airdry.
7. Apply PAP pen and continue with the procedure from the Milk block (ie do not block endogenous peroxidase).




1. Block endogeneous peroxidase                 2 min
2. Rinse well in tap water             
3. Rinse well in buffer wash solution
4. Block non-specific binding                   3 min
6. Apply Localisation antibody and negative control reagent and incubate at room temp for 15 min
7. Rinse well in buffer wash solution
8. Link reagent (yellow label)                  10 min
9. Rinse well in buffer wash solution
10. Streptavidin reagent (red label)                    10 min
11. Rinse well in buffer wash solution
12. DAB reagent                                 6 min
13. Rinse well in tap water
14. Nuclear staining with Harris's haematoxylin 2-3 min
15. Differentiate, blue, dehydrate, clear and mount in usual manner.

Henwood A F, (1981) Aust J Med Lab SC, 2:55-66.
Henwood A F, (1982) Aust J Med Lab SC, 3:21-27.
Henwood,A.F., (1991) Aust J Med Lab Sc 12:59-61.

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