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 journalArticlepeer-review

14 Scopus citations


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
Issue number4
StatePublished - Dec 2002


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


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