Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections: androgen receptor, estrogen receptor, progesterone receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen revealed by antigen retrieval techniques

Hum Pathol. 1994 Mar;25(3):263-70. doi: 10.1016/0046-8177(94)90198-8.


Different variations of the antigen retrieval technique using different retrieval solutions have been evaluated for their effectiveness in restoring the antigenicity of six intranuclear antigens, each of which is a potentially valuable prognostic indicator in formalin-fixed, paraffin-embedded tissue sections. The results of immunohistochemical staining for estrogen receptor, progesterone receptor, androgen receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen were compared following the different antigen retrieval approaches. The strongest immunostaining signal with the clearest background was obtained by microwave heating of dewaxed paraffin sections for 10 minutes in 0.05 mol/L glycine HCl (pH 3.5) or in citrate buffer solution (pH 6). Urea solution, distilled water, and lead thiocyanate solution yielded improvements with some antigens, but less consistently and less impressively than glycine HCl buffer or citrate buffer. Following antigen retrieval nuclear staining was sharply defined and could be achieved consistently in a variety of tissues after formalin fixation for as long as 7 days. The duration of fixation, however, was an important variable; generally, the longer the fixation time the more vigorous the retrieval procedure required. This study demonstrates the ability to stain a variety of intranuclear antigens, which are not readily demonstrable otherwise, in formalin-paraffin sections with a high degree of consistency and reproducibility. The availability of methods that are effective in paraffin sections may facilitate studies of the possible value of these markers as prognostic indicators for predicting the response of major tumors to different forms of therapy. This study also provided insight into the basic principles of the antigen retrieval method, which may be helpful in attempts to develop a more uniformly standardized technique applicable to many different antigen systems.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Neoplasm / analysis*
  • Biomarkers, Tumor / analysis*
  • Cell Nucleus / chemistry
  • Cell Nucleus / ultrastructure
  • Formaldehyde*
  • Glycine
  • Humans
  • Immunohistochemistry / methods*
  • Ki-67 Antigen
  • Microwaves
  • Neoplasm Proteins / analysis
  • Neoplasms / chemistry
  • Neoplasms / diagnosis
  • Neoplasms / pathology
  • Nuclear Proteins / analysis
  • Paraffin Embedding*
  • Prognosis
  • Proliferating Cell Nuclear Antigen
  • Receptors, Androgen / analysis
  • Receptors, Estrogen / analysis
  • Receptors, Progesterone / analysis
  • Staining and Labeling / methods
  • Thiocyanates
  • Tumor Suppressor Protein p53 / analysis
  • Urea


  • Antigens, Neoplasm
  • Biomarkers, Tumor
  • Ki-67 Antigen
  • Neoplasm Proteins
  • Nuclear Proteins
  • Proliferating Cell Nuclear Antigen
  • Receptors, Androgen
  • Receptors, Estrogen
  • Receptors, Progesterone
  • Thiocyanates
  • Tumor Suppressor Protein p53
  • Formaldehyde
  • Urea
  • Glycine