Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

Author(s): Shi SR, Key ME, Kalra KL

Publication: J Histochem Cytochem, 1991, Vol. 39, Page 741-8

PubMed ID: 1709656 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to evaluate different antigen retrieval solutions, and microwave and conventional oven heating regimes for the analysis of FFPE specimens.

   Summary of Findings:

   The authors report that the following conditions resulted in optimal immunostaining while minimizing background staining: microwave heating of sections in a saturated lead thiocyanate solution for two five-minute cycles separated by one minute. Microwave heating was superior to heating in a conventional oven particularly for tissue that had been formalin fixed for 24 hours or longer. Lead thiocyanate proved slightly more efficacious than 1% zinc sulfate and significantly better than distilled water. Enzyme pre-digestion in a trypsin solution was not necessary for optimal antigen retrieval. Immunoreactivity of two representative antibodies (vimentin, pan-cytokeratin) was also enhanced in specimens that were stored in formalin for prolonged periods of time (2 wk-2 yr).

   Biospecimens

   • Tissue - Tonsil
   • Tissue - Lung

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Antigen retrieval	Saturated lead thiocyanate 1% Zinc sulfate Distilled water
   Analyte Extraction and Purification	Temperature of heat-induced retrieval	Microwave oven heating, 5 min Microwave oven heating, 10 min Microwave oven heating, 2 X 5 min Conventional oven heating, 5 min Conventional oven heating, 10 min Conventional oven heating, 2 X 5 min
   Analyte Extraction and Purification	Protein digestion	0.1% Trypsin, 30 min, 37 degrees C No trypsin incubation
   Biospecimen Preservation	Time in fixative	24 h - 2 yr

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2. Study Purpose

   The purpose of this study was to evaluate microwave-mediated antigen retrieval in differentially fixed tissues. Two fixatives (formalin, ethanol) and three fixation temperatures were evaluated at a constant fixation duration of 22 h.

   Summary of Findings:

   The deleterious effects on immunostaining of a formalin sensitive antigen observed at higher fixation temperatures (25 and 37 degrees C) were attenuated by microwave-mediated antigen retrieval with lead thiocyanate. Heat-induced antigen retrieval, while extremely effective for FFPE tissues, did not alter immunostaining of tissues preserved in ethanol.

   Biospecimens

   • Tissue - Skin

   Preservative Types

   • Formalin
   • Ethanol

   Diagnoses:

   • Neoplastic - Melanoma
   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Temperature of fixation/preservation	4 degrees C 25 degrees C 37 degrees C
   Biospecimen Preservation	Type of fixation/preservation	Ethanol Formalin (buffered)

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