A modified reduced-temperature antigen retrieval protocol effective for use with a polyclonal antibody to cyclooxygenase-2 (PG 27)

Shan Rong Shi, Richard J Cote, Cheng Liu, Mimi C. Yu, Jose Esteban Castelao, Ronald K. Ross, Clive R. Taylor

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Antigen retrieval is now a standard procedure in immunohistochemical studies of tissues for diagnosis and research. While the most commonly used protocol (20 minutes at 100°C in citrate buffer pH 6.0) is effective for many antibody/antigen combinations, experience has shown that in some instances, this standard approach fails. Under these circumstances, a successful antigen retrieval protocol may still be established by varying key conditions in the antigen retrieval process. The authors previously have advocated a test battery approach to determine the optimal conditions for antigen retrieval, illustrated here with respect to a polyclonal antibody to cyclooxygenase-2 (PG-27) that failed to give a positive staining result after orthodox antigen retrieval. The key feature of this modified antigen retrieval protocol is heating the deparaffinized tissue sections at a reduced temperature (90°C as opposed to 100°C). For this particular antibody, a boiling condition yields a negative result, a principal reason why previous investigators have used a tyramide signal amplification system to achieve satisfactory immunohistochemical results with this antibody. The optimal antigen retrieval protocol established in the authors' laboratory for this polyclonal antibody to cyclooxygenase-2 (PG-27) was evaluated in a study of formalin-fixed, paraffin-embedded cell lines and 31 bladder cancer tissue blocks using the tissue microarray technique, with side-by-side comparison between the results obtained by a tyramide signal amplification method (without antigen retrieval) and a standard immunohistochemical method with the optimized antigen retrieval protocol. The reduced temperature antigen retrieval protocol yielded a comparable or superior immunostaining for cyclooxygenase-2 both in cell lines and tissue blocks. In conclusion, use of the test battery approach allowed development of a modified antigen retrieval technique that provides a more reliable, much simpler approach for the demonstration of cyclooxygenase-2 in archival tissues.

Original languageEnglish
Pages (from-to)368-373
Number of pages6
JournalApplied Immunohistochemistry and Molecular Morphology
Volume10
Issue number4
DOIs
StatePublished - Dec 1 2002
Externally publishedYes

Fingerprint

Cyclooxygenase 2
Antigens
Temperature
Antibodies
Cell Line
Urinary Bladder Neoplasms
Citric Acid
Paraffin
Heating
Formaldehyde
Buffers
Research Personnel
Staining and Labeling

Keywords

  • Antigen retrieval
  • Cyclooxygenase-2
  • Formalin-fixed
  • Immunohistochemistry
  • Paraffin-embedded tissue

ASJC Scopus subject areas

  • Anatomy
  • Medical Laboratory Technology

Cite this

A modified reduced-temperature antigen retrieval protocol effective for use with a polyclonal antibody to cyclooxygenase-2 (PG 27). / Shi, Shan Rong; Cote, Richard J; Liu, Cheng; Yu, Mimi C.; Castelao, Jose Esteban; Ross, Ronald K.; Taylor, Clive R.

In: Applied Immunohistochemistry and Molecular Morphology, Vol. 10, No. 4, 01.12.2002, p. 368-373.

Research output: Contribution to journalArticle

Shi, Shan Rong ; Cote, Richard J ; Liu, Cheng ; Yu, Mimi C. ; Castelao, Jose Esteban ; Ross, Ronald K. ; Taylor, Clive R. / A modified reduced-temperature antigen retrieval protocol effective for use with a polyclonal antibody to cyclooxygenase-2 (PG 27). In: Applied Immunohistochemistry and Molecular Morphology. 2002 ; Vol. 10, No. 4. pp. 368-373.
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