Fig. 3: MJF-14 PLA stains considerable pathology in DLB brains but is not sensitive to Lewy bodies.

a Technical negative controls: PLA –ligase in the reaction (left) and PLA –antibody (right) display significant amounts of red channel signal when autofluorescence is not quenched. Scale bars = 20 µm. b TrueBlack-quenching of autofluorescence effectively removes background signal in technical negative controls (here, PLA –antibody). Scale bar = 20 µm. c Representative images of sections immunostained with MJF-14 PLA (red), pS129-α-synuclein (pS129, grey), and DAPI nuclear stain (blue) in control (left) and DLB (right). Examples of PLA-positive neurons are indicated with arrows, while a LB-positive neuron is indicated by an arrowhead. Scale bars = 20 µm. d Quantification of PLA particle area in control and DLB, compared by a non-parametric Mann–Whitney U-test. Graph displays mean ± SEM of the total PLA area in µm²/image, with each dot representing one image. ****p < 0.0001. e Close-up z-stack rendering of a LB-containing neuron, with (left) and without (right) pS129, displaying the lack of PLA-staining of the LB. PLA-signal (red, arrows) and LB (grey, arrowhead) is highlighted. Smaller panels on the right show weak pS129 IF-labelling of PLA particles.