title: A modified reduced-temperature antigen retrieval protocol effective for use with a polyclonal antibody to cyclooxygenase-2 (PG 27).
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 degrees 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 degrees C as opposed to 100 degrees 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.
authors.affiliation: Department of Pathology, University of Southern California Keck School of Medicine, Los Angeles, California 90033, USA.
authors.forename: Shan-Rong
authors.initials: SR
authors.lastname: Shi
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Cote
authors.forename: Cheng
authors.initials: C
authors.lastname: Liu
authors.forename: Mimi C
authors.initials: MC
authors.lastname: Yu
authors.forename: Jose Esteban
authors.initials: JE
authors.lastname: Castelao
authors.forename: Ronald K
authors.initials: RK
authors.lastname: Ross
authors.forename: Clive R
authors.initials: CR
authors.lastname: Taylor
mesh_terms: D000906:Antibodies; D000941:Antigens / Q000378:metabolism*; D001943:Breast Neoplasms / Q000201:enzymology / Q000473:pathology; D051546:Cyclooxygenase 2; D005260:Female; D005557:Formaldehyde; D006358:Hot Temperature; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D007527:Isoenzymes / Q000276:immunology* / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins; D016612:Paraffin Embedding; D011451:Prostaglandin-Endoperoxide Synthases / Q000276:immunology* / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000473:pathology; D016707:Tissue Fixation; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000201:enzymology / Q000473:pathology
chemical_list: D000906:Antibodies; D000941:Antigens; D007527:Isoenzymes; D008565:Membrane Proteins; D005557:Formaldehyde; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases