Detection of proliferating cell nuclear antigen in paraffin-embedded specimens is dependent on preembedding tissue handling and fixation.
Immunohistochemical detection of proliferating cell nuclear antigen (PCNA), a cell cycle-related protein used to estimate tumor growth fraction, is variable in formalin-fixed compared with methanol-fixed tissue specimens. This is assumed to result from conformational changes in the antigenic epitope induced by formaldehyde; therefore, to be susceptible to retrieval in archival specimens. In this study, formalin fixation reduced the intensity of staining and the number of positive cells to approximately 25% of those in methanol-fixed material. The washing of tissue specimens prior to methacarn fixation also reduced PCNA staining. Loss of staining was not restored after use of a commercial retrieval kit recommended for PCNA immunohistochemistry. Immunoblotting of formalin fixatives and saline washings after removal of tissue specimens consistently demonstrated the presence of PCNA-like activity in solution. We conclude that the exceptional solubility of PCNA is responsible for reduced immunostaining in formalin-fixed material, that the loss is irreversible, and that methanol or methacarn is the fixative of choice for PCNA immunohistochemistry.
Burford-Mason
A P
AP
Conacher Laryngeal Laboratory, Toronto Hospital, Ontario, Canada.
MacKay
A J
AJ
Cummins
M
M
Dardick
I
I
D000818:Animals; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D016612:Paraffin Embedding; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis*; D051381:Rats; D013048:Specimen Handling / Q000379:methods*; D013194:Staining and Labeling; D016707:Tissue Fixation
D018809:Proliferating Cell Nuclear Antigen