Immunohistochemistry for alpha-synuclein has become the histological technique of choice for the diagnosis for Parkinson's disease, Dementia with Lewy bodies and Multiple System Atrophy (www.ICDNS.org). Nevertheless, no standardised protocol has been proposed. We have reviewed 242 of the 270 studies published until June 2005 that mentioned immunohistochemistry for anti-alpha synuclein on human tissue and we found that only 75 (31%) used commercial antibodies. We also noted that protocols, particularly dilution and antigen unmasking, varied between studies, even when the same antibody was employed. In order to establish a standardised protocol for alpha-synuclein immunohistochemistry, which can be applied in diagnostic neuropathology we tested seven commercial monoclonal antibodies in brains of subjects with Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, multiple sclerosis with incidental Lewy bodies and aged-matched normal brain and determined for each antibody the best suited protocol for antigen unmasking. We evaluated the intensity of immunolabelling in Lewy bodies, neuropil threads, dendrites, presynaptic terminals, granular cytoplasmic positivity, peri-axonal positivity, glial inclusions and non-specific immunolabelling. Although our results showed that all the antibodies detected alpha-synuclein inclusions, differences were noted between antibodies, particularly with regard to the detection of glial inclusions. From our study, the best antibodies of the seven tested appeared to be those directed against amino acids 116-131 and 15-123 and we suggest them to be used in routine diagnostic practice for alpha-synucleinopathies. Keywords : alpha-synuclein ; immunohistochemistryAlpha-synuclein (αSN) is a highly conserved, natively unfolded 18 kDa protein [1] which represents the major component of Lewy bodies (LBs) [2], and the glial cytoplasmic inclusions (GCIs) associated with Multiple System Atrophy (MSA) [3]. The increased sensitivity of αSN immunohistochemistry over conventional histological stains has established the technique as a standard tool in the diagnosis of Parkinson's disease (PD), Dementia with Lewy Bodies (DLB) and MSA [4], and led to new insights regarding the extent and variation of αSN-containing structures and their formation. αSN aggregations vary throughout the brain, with brainstem pathology characterized by compact LBs and neuropil threads. Pigmented neurones of the substantia nigra may also contain diffuse fine or coarse granules, which likely represent the state of aggregation preceding LBs [5,6]. Cortical pathology consists of more diffuse LBs and punctate granular positivity that corresponds to pre-synaptic terminals [7].Despite the growing frequency with which immunohistochemistry is used as a diagnostic tool for PD, DLB and MSA, a standardized protocol for αSN has not been established. Pretreatment with proteinase K or formic acid has been reported to increase the immunoreactivity of αSN; however, the only studies to investigate this systematically ...
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