Development of an optimal protocol for antigen retrieval: a 'test battery' approach exemplified with reference to the staining of retinoblastoma protein (pRB) in formalin-fixed paraffin sections

J Pathol. 1996 Jul;179(3):347-52. doi: 10.1002/(SICI)1096-9896(199607)179:3<347::AID-PATH559>3.0.CO;2-L.


The retinoblastoma (RB) gene, which encodes the nuclear RB protein (pRB), is believed to be involved in cell cycle control and cell differentiation. Studies have demonstrated that loss of RB function may play a role in tumour formation and progression of a variety of human tumours, such as bladder, lung, breast, and prostate cancers. The immunohistochemical detection of pRB expression in formalin-paraffin sections of human cancer has potential advantages of convenience, economy, and compatibility with routine surgical pathology practice. In practice, however, results using pRB antibodies on routinely processed, paraffin-embedded tissue have been inconsistent. In this study, the antigen retrieval (AR) method has been applied to the immunohistochemical detection of pRB in paraffin-embedded tissues and a 'test battery' approach has been developed to identify the principal variables that result in the optimal AR protocol. This approach includes the use of buffered solutions at pH 1, 6, and 10 with three different heating conditions (temperatures 120 degrees C, 100 degrees C, and 90 degrees C). In the example described here with antibody RB-WL-1, the low pH solution with the microwave heating at 100 degrees C proved most effective. Both fresh and routinely processed formalin-paraffin tissues of normal and bladder carcinoma were used for a comparison of the pRB immunostaining. The AR method was evaluated by comparing the immunohistochemical staining result on routinely processed formalin-paraffin sections with frozen sections of the same tumour. A consistent intensity of immunohistochemical staining for pRB was achieved using the identified optimal AR protocol on formalin-paraffin sections. All slides showed positive staining of pRB in normal mesenchymal and epithelial tissues. The pattern of pRB localization and intensity of staining was similar to that obtained in frozen sections, though the intensity obtained by AR treatment on paraffin sections was slightly to moderately stronger than that obtained in frozen sections. Once the protocol was identified, it was tested using routinely processed paraffin tissue sections of 245 cases of bladder carcinoma, with consistent pRB immunostaining results. The protocol described is simple to perform and gives reproducible results for evaluation of pRB expression by immunohistochemistry.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cryopreservation
  • Formaldehyde
  • Humans
  • Hydrogen-Ion Concentration
  • Immunoenzyme Techniques*
  • Microwaves
  • Neoplasm Proteins / metabolism*
  • Paraffin Embedding
  • Retinoblastoma Protein / metabolism*
  • Time Factors
  • Urinary Bladder Neoplasms / metabolism


  • Neoplasm Proteins
  • Retinoblastoma Protein
  • Formaldehyde