Demonstration of pathological prion protein accumulation in the central nervous system is required to establish the diagnosis of transmissible subacute encephalopathies. In humans, this is frequently achieved using prion protein immunohistochemistry in paraffin-embedded tissue, a technique that requires multiple epitope retrieval and denaturing pretreatments. In addition to being time-consuming, this procedure induces tissue alterations that preclude accurate morphological examination. The aim of this study was to simplify prion protein immunohistochemistry procedure in human tissue, together with increased sensitivity and specificity. We screened a panel of 50 monoclonal antibodies produced using various immunogens (human and ovine recombinant prion protein, prion protein peptides, denatured scrapie-associated fibrils from 263K-infected Syrian hamsters) and directed against different epitopes along the human prion protein sequence. A panel of different forms of genetic, infectious and sporadic transmissible subacute encephalopathies was assessed. The monoclonal 12F10 antibody provided a high specificity and fast immunodiagnosis with very limited denaturing pretreatments. A standardized and reliable fast immunostaining procedure was established using an automated diagnostic system (Nexes, Ventana Medical Systems) and allowed prion protein detection in the central nervous system and in tonsil biopsies. It was evaluated in a series of 300 patients with a suspected diagnosis of transmissible subacute encephalopathies and showed high sensitivity and specificity.