Pathologic diagnosis of Lewy Body disease describes 4 stages of distribution of α-synuclein in the brain based on algorithm and neuropathologist's opinion. Sections (6 μm) of paraffin-embedded brain tissue (from midfrontal, midtemporal, inferior parietal, anterior cingulate, entorhinal cortices, amygdala and midbrain) were stained for α-synuclein immunostain (Zymed; 1:50). Immunohistochemistry was performed using the VECTASTAIN ABC method with alkaline phosphatase as the colour developer. McKeith criteria (McKeith et al., 1996) were modified to assess the following categories of Lewy body disease:

Nigral Lewy bodies were identified as round, intracytoplasmic structures with a darker halo. In the cortex, Lewy bodies were identified as round intracytoplasmic structures, often lacking any halo and with an eccentric nucleus. Only intracytoplasmic Lewy bodies were used as an indicator of positive staining.

Note: Both limbic type and neocortical lewy body disease - are considered "cortical" Lewy body disease; however in our study (and others) only neocortical are related to dementia.