Antigen retrieval in prion protein immunohistochemistry

Transmissible spongiform encephalopathies are a group of neurodegenerative diseases occurring in both humans and animals and are most likely caused by prions. Neuropathological confirmation of the clinical diagnosis has been a problem because of the difficulty in epitope retrieval from formalin-fixe d, paraffin-embedded brain specimens. Many different protocols for the dete ction of prions in brain tissue have been used. Thus far, picric and/or for mic acid, steam autoclaving at 121C of sections, microwave treatment, and 4 M guanidine thiocyanate treatment have been suggested. The objective of ou r experiment was to obtain the standard pretreatment(s) resulting in optima l immunostaining. In the experiment, successive tissue slides of brain spec imens of several Creutzfeldt-Jakob disease and control patients were staine d using different combinations of pretreatments. Using densitometric analys is, several well-defined locations per section were examined and prion immu nostaining was quantified. The results showed that autoclaving is necessary for antigen retrieval and cannot be substituted by microwave treatment. Th e best results were obtained when the following combination was used in the specified order: 15 min saturated picric acid, 10 min steam autoclaving at 121C, 5 min 88% formic acid, and 2 hr 4 M guanidine thiocyanate at 4C.