Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies.

Author(s): Shi SR, Imam SA, Young L, Cote RJ, Taylor CR

Publication: J Histochem Cytochem, 1995, Vol. 43, Page 193

PubMed ID: 7822775 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to evaluate the effects of different heat-induced antigen-retrieval buffers under different pH conditions on the immunostaining of monoclonal antibodies to cytoplasmic antigens (AE1, HMB45, NSE), nuclear antigens (MIB-1, PCNA, ER), and cell surface antigens (MT1, L26, EMA) in FFPE sections. An unspecified number of tonsil, pancreas, lymph nodes with metastatic squamous cell carcinoma, melanoma, and breast carcinoma were fixed in 10% formalin for 24 h and paraffin embedded. Five µm sections were dried for an hour at 60˚C, dewaxed, rehydrated and endogenous peroxidase was blocked using unspecified methods. Slides were placed in jars containing the 7 different antigen retrieval solutions tested prepared at pH 1-10 depending on solution and microwaved for 5 +5 min at 2450 mHz and 600 W. When required more antigen retrieval solution was added between microwave steps. After microwaving slides remained in antigen retrieval solution for 15 min before washing and immunostaining with the different antibodies. Immunostaining of each slide was scored at extremely strong, strong, moderate, weak or negative by three blinded authors independently..

   Summary of Findings:

   The authors report that pH had a greater influence on immunostaining than buffer solution; no significant differences were seen in maximal staining achieved with each different buffer. The influence of pH fell into three categories: antibodies with no significant variation correlated to pH (including L26, PCNA, AE1, EMA, NSE); antibodies that stained well in the very low and high pH ranges, but stained poorly under moderately acidic conditions (pH 3-6; including MIB-1, ER)); and antibodies for which staining improved as pH increased (MT1, HMB45). They concluded that, although heating is the most important factor in antigen retrieval, pH of the retrieval solution may also play a role and should be optimized for the antibody of interest. The damage to or separation of the tissue that is occasionally noted when heating a strongly alkaline solution was circumvented with Tris-HCl and sodium acetate buffers at a pH of 9, which adequately preserved morphology.

   Biospecimens

   • Tissue - Lymph Node
   • Tissue - Breast
   • Tissue - Skin
   • Tissue - Tonsil
   • Tissue - Pancreas

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Not specified
   • Neoplastic - Melanoma

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	pH	2.0 - 9.4
   Analyte Extraction and Purification	Antigen retrieval	Acetate Citrate Phosphate Tris-HCl Barbitol sodium/sodium acetate Sodium phosphate/citric acid Dimethyl glutaric acid/NaOH Distilled water

   View more