New pretreatment method for
immunohistochemistry for abnormal prion protein
Kensuke Sasaki\ Katsumi Doh-ura^ and Toru Iwaki^
^Department of Neuropathology, Neurological Institute, Kyushu Univer- sity, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 Japan ^Department of Prion Research, Tohoku University School of Medicine
<e-mail> ksasaki@np.med.kyushu-u.ac.jp
Abstract
Immunohistochemistry for prion protein (PrP) is essential for pathological diagnosis of prion diseases, however, certain pretreatment procedures are required for antigen retrieval of formalin-fixed paraffin-embedded sec- tions. Although hydrolytic autoclaving and formic-acid treatment are usually employed, these methods tend to result in nonspecific high back- ground staining and poorly preserved sections. Thus, we tried autoclav- ing of the sections with nonionic detergent as follows: autoclave sections with 0.1% Triton X-100 in 50mM Tris-HCl buffer pH7.6, 12PC 20 min- utes. Both monoclonal antibody (clone 3F4) and polyclonal anti-PrP-C-terminal antibody were used for human cases with Creutzfeldt-Jakob disease and mouse models of transmissible spongiform encephalopathies. This detergent autoclaving method resulted in signifi- cantly higher signal/noise ratio for accumulated PrP in the follicular den- dritic cells compared to conventional pretreatment methods. Although synaptic staining in the brain was obtained rather weakly than with con- ventional methods, pretreatment with detergent autoclaving contributed to lower background and well preserved sections so that we could recognize PrP deposition well. Normal-form cellular PrP was not immunostained in control cases. These results were observed for both monoclonal and polyclonal antibodies, and both human and murine materials. The lower background staining, obtained with detergent autoclaving, enabled double immunofluorescence efficiently on the same sections. We, thus, propose the detergent autoclaving method as a useful choice of the pretreatment for PrP immunohistochemistry
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