Antigen retrieval in formalin-fixed tissues using microwave energy

• US 5,244,787 A
• Filed: 01/31/1991
• Issued: 09/14/1993
• Est. Priority Date: 01/31/1991
• Status: Expired due to Term

• Current Assignee: Biogenex Laboratories Incorporated
• Original Assignee: Biogenex Laboratories Incorporated
• Inventors: Key, Marc E., Shi, Shan-Rong, Kalra, Krishan L.
• Primary Examiner(s): Rosen, Sam
• Application Number:
  US07/649,036
• Time in Patent Office: 957 Days
• Field of Search: 424/3, 435/7.9, 435/7.94, 436/518
• US Class Current: 435/7.9
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 1/44 Sample treatment involving radiation, e.g. heat
• CPC Class Codes: G01N 2474/20 Immunohistochemistry assay
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Abstract

A method of immunologically staining a formalin-fixed tissue preparation, which comprises (a) subjecting a formalin-fixed tissue preparation to microwave energy while the tissue preparation is submersed in water for a time sufficient to increase immunostaining efficiency; (b) removing the tissue preparation from the water and cooling; and (c) contacting the tissue preparation with an immunological staining reagent.

Claims

1. A method for the preparation of formalin-fixed tissue for immunological staining, which comprises:

heating a formalin-fixed tissue preparation submersed in water with microwave energy for a time sufficient to increase immunostaining of said preparation in relation to immunostaining in the absence of said heating.

2. The method of claim 1, wherein said time is sufficient to boil said water.

3. The method of claim 1, wherein said water is an aqueous solution of a metal salt.

4. The method of claim 3, wherein said metal salt is a salt of lead or zinc.

5. The method of claim 2, wherein said water contains a dissolved metal salt and boils at a temperature at or above 100°

C.

6. The method of claim 1, wherein said microwave energy has a frequency of from 1 to 50 GHz.

7. The method of claim 2, wherein said microwave energy is applied at a rate sufficient to cause 50 ml of said water to increase in temperature at a rate of from 0.5°

to 5.0°

C. per second prior to boiling.

8. The method of claim 2, wherein said boiling continues for up to 20 minutes.

9. The method of claim 3, wherein said method further comprises removing said tissue preparation from said water after being subjected to said microwave energy and washing to remove said aqueous solution.

10. A method of immunologically staining a formalin-fixed tissue preparation, which comprises:

(a) subjecting a formalin-fixed tissue preparation or tissue section on a microscope slide to microwave energy while said tissue preparation is submersed in water for a time sufficient to boil said water;

(b) removing said tissue preparation from said water and cooling to a temperature below 100°

C.; and

(c) contacting said tissue preparation with an immunological staining reagent or series of reagents.

11. The method of claim 10, wherein said water is an aqueous solution of a metal salt.

12. The method of claim 11, wherein said metal salt is a salt of lead or zinc.

13. The method of claim 11, wherein said water contains a dissolved metal salt and boils at a temperature above 100°

C.

14. The method of claim 10, wherein said microwave energy has a frequency of from 1 to 50 MHz.

15. The method of claim 10, wherein said microwave energy is applied at a rate sufficient to cause 50 ml of said water to increase in temperature at a rate of from 0.5°

to 5°

C. per second prior to boiling.

16. The method of claim 10, wherein said boiling continues for up to 20 minutes.

17. The method of claim 10, wherein said tissue preparation is stained with an enzyme-labelled monoclonal antibody.

18. The method of claim 10, wherein said tissue preparation is first reacted with an unlabeled monoclonal or polyclonal antibody and subsequently reacted with a reagent or series of reagents to introduce an enzyme label at the site of the antigen-antibody reaction.