Creutzfeldt-Jakob disease: Carnoy's fixative improves the immunohistochemistry of the proteinase K-resistant prion protein

The neuropathological diagnosis of Creutzfeldt-Jakob disease relies on the immunohistochemical demonstration of the proteinase-K resistant form of the prion protein (PrPres) in the brain tissue. The antigenicity of PrPres is strongly reduced by the formalin solution widely used to fix the tissue, th us the PrPres immunoreactivity is inconsistently detectable in formalin-fix ed tissue. A better PrPres immunostaining can be obtained by using Carnoy's fixing solution, which is composed of ethanol, chloroform and acetic acid (6:3:1). PrPres can easily be extracted from Carnoy's-fixed, paraplast-embe dded tissue, Accordingly, Carnoy's-fixed tissue can prior to immunolabeling be subjected to proteinase K and guanidine thiocyanate, which respectively eliminate the normal cellular form of prion protein and promote protein de naturation, In comparison with the best protocols for formalin-fixed tissue (i.e. - hydrolytic autoclaving or autoclaving in distilled water followed by formic acid and guanidine thiocyanate), PrPres immunostaining carried ou t on sections cut from Carnoy's-fixed, paraplast-embedded tissue blocks and subjected to proteinase K and guanidine thiocyanate, proved more successfu l to detect and map both diffuse and focal PrPres immunoreactivity, and to correlate the immunoreactivity pattern with MV polymorphism at PRNP codon 1 29 and PrPres banding and glycosylation pattern revealed by Western blot.