Acta Neuropathol (2009) 118:659–671 Materials and methods Case and tissue selection This study was based on 225 cases with an established diagnosis of sCJD obtained after clinical, neuropathologi- cal, and molecular examination. Cases of genetic and acquired forms of CJD were excluded. The study was restricted to cases from which large amounts of both frozen and ﬁxed brain tissue were available. 200 patients were obtained from a group of *240 consecutive cases referred for diagnosis (*40 cases lacked sufﬁcient frozen tissue), whereas the remaining 25 were speciﬁcally chosen to increase the number of cases with mixed phenotypes. More precisely, the 25 additional cases were selected based on the demonstration of a mixed synaptic and perivacuolar pattern of PrP deposition by PrP immunohistochemistry (see ‘‘Results’’). 61 patients died in the USA between 1990 and 2001, and 164 in Europe (124 in Italy, 34 in Germany, and 6 in Belgium) between 1993 and 2007. Brains were removed at autopsy and either one half or selected coronal sections of tissue, including all major brain structures and nuclei, were immediately frozen and stored at -80 C. The remaining tissue was ﬁxed in for- malin and was used for neuropathological examination and PrP immunohistochemistry. Samples of frozen gray matter (between 50 and 100 mg) for protein analysis were obtained from the following regions: frontal (superior and middle frontal gyri), temporal (superior and middle tem- poral gyri), parietal (inferior parietal lobule), and occipital (calcarine cortex and lateral occipital gyrus) cortices, hip- pocampus (Ammon’s horn), limbic cortices (entorhinal cortex anterior, insular cortex, and cingulate gyrus), stria- tum (caudate and putamen nuclei), thalamus (medial and lateral nuclei), hypothalamus, brainstem (midbrain peri- aqueductal gray, pontine periaqueductal gray including locus coeruleus, medullar periventricular gray), cerebellum (hemisphere and vermis). The parietal cortex and the 3 samples from the brainstem were not available in 30 cases from Germany. Furthermore, 1 or 2 samples were occa- sionally lacking in some other cases [altogether 34 samples, mostly from the hypothalamus (n = 15) and amygdala (n = 11)]. Sampling of frozen tissues was per- formed by the same investigator (PP) according to a deﬁned protocol across the whole series of cases. Blocks of ﬁxed tissue were taken from the opposite half of the brain and were used for histopathologic examination. Sampling of ﬁxed tissues was performed by the same investigator (PP) according to a deﬁned protocol in 160 cases. The remaining 65 cases were sampled in Munich (Germany) and Indianapolis (USA). In these Centers, which are both involved in CJD National surveillance and Brain banking, CJD brains are sampled extensively according to protocols, 661 which included all the areas that were of interest for this study. Sampling of both ﬁxed and frozen tissue was per- formed twice in a subgroup of 10 codon 129 MM subjects. Protein analysis Preparation of samples, Western blotting (WB), and PrPSc typing were performed according to established methods [25, 26, 33]. In particular, for the sensitive detection of PrPSc types 1 and 2 concurrence, we followed the method of Notari et al. [25]. All samples were homogenized in lysis buffer plus at pH 6.9, digested with proteinase K (PK) (Roche Diagnostics, speciﬁc activity by certiﬁcate of analysis: 47.9 U/mg) at a ﬁnal concentration of 10 U/mL and run in a 6.5 cm long separating gel. In addition, selected samples from MV2 cases with kuru plaques (MV 2K) and MM or MV subjects showing mixed pathological features but only PrPSc type 1 after the run in the 6.5 cm long gel were also run in a 15 cm long gel. To this aim, at least one sample from each cortical lobe was re-analyzed. All immunoblots were probed with the monoclonal anti- body 3F4 with epitope at PrP residues 108-111 at 0.1 lg/ ml concentration (Signet Labs, MA, USA). In addition, immunoblots were probed with the monoclonal antibody 1E4 with epitope mapped at PrP residues 98–100 at 2 lg/ ml concentration (Sanquin Reagents, Amsterdam, The Netherlands) in the following cases: (a) all samples from VV1 cases; (b) 20 neocortical samples from 10 MM1 cases with pure synaptic PrP deposition, and (c) all samples from 30 MM or MV cases, which showed a mixed pattern of PrP deposition by immunohistochemistry and either no evi- dence or a very scanty and focal (1 or 2 areas) type 2 accumulation after immunoblotting with the 3F4 antibody. Finally, selected blots from MV 2K samples were also probed with SAF60 against PrP residues 157–161, at 0.4 lg/ml. We quantitatively calculated the ratio between type 1 and type 2 in the non-glycosylated PrPSc after densito- metric analyses of immunoblots probed with 3F4 using the AIDA Image Analyzer software (RayTest). This choice was in part driven by our experience indicating that deglycosylation is associated to a loss of deﬁnition in the type 1 (21 kDa) and type 2 (19 kDa) bands which makes their distinction and relative quantitation less reliable. Furthermore, given that it has been previously shown [28, 30] that the relative abundance of glycosylated versus non- glycosylated PrPSc shows only relatively minor differences among the sCJD subtypes associated to either PrPSc type 1 or PrPSc type 2, we assumed that the analyses of the unglycosylated form would be representative of the whole protein. We tested this hypothesis by comparing densito- metric analyses of non-glycosylated glycoforms versus deglycosylated preparations from representative regions of 123