Fig. 3

Quantification of nuclear aSyn pathology in cases of dementia with lewy bodies in isolated nuclei. a Representative confocal micrograph images of nuclei isolated from lateral temporal cortex (× 40 objective, inserts ii + iv taken with 10 × digital zoom, scale = 5 µm) stained for pan-aSyn (Syn-1, i + ii) and phospho-serine 129 aSyn (pS129, iii + iv). b Optimisation of immunoblot detection of aSyn from cytoplasmic (c) and nuclear (n) fractionates generated from control (Con) and dementia with Lewy bodies (DLB) cases. Comparisons of post-transfer membrane phosphate buffered saline heating (5 min boiling) or 4% paraformaldehyde chemical (30 min 4% paraformaldehyde) fixation with non-fixed (untreated) controls is shown, captured under optimal and overexposed settings. Monomeric (mono) and oligomeric (oligo) immunoreactivity is highlighted alongside a ranking of fixation methods-based retention of the aSyn species (+ = enhanced and++ = very enhanced). Quantification of c pan-aSyn via Syn-1, and d pS129 via EP1536Y, (i) example immunoblots following boiling fixation are shown at optimised and overexposed capture settings and mono, oligo and truncated (trunc) species are highlighted (i). Loading controls of GAPDH and Histone H3 demonstrate fractionation purity and resulting immunoreactivity from similarly fractionated aSyn knockout mouse brain tissue (aSyn^−/−) demonstrated antibody specificity. Quantification of nuclear aSyn monomers between DLB and controls cases is also shown (ii), demonstrating increased pS129 Syn in DLB cases compared to controls (n = 5 per group), despite no change in total aSyn levels (n = 9 and 8 for con and DLB respectively) and no difference in the nuclear: cytoplasmic ratio of total aSyn (c iii). Data shown as scatter plots, expressed relative to mean control values (Rel. Con) with mean ± SEM also indicated, ** = p < 0.01