Z2 axioimager

Using a protocol adapted from Maric et al. [36 (link)] and Murray et al. [37 (link)], paraffin-embedded tissue microarray sections of AD and normal middle temporal gyrus were processed as above. Imaging was carried out with an automated fluorescence microscope (Zeiss Z2 Axioimager) equipped with MetaSystems VSlide slide scanner (MetaSystems) running MetaFer (V 3.12.1) with a 20 × air objective (0.9 NA). This microscope is equipped with 6 custom excitation/dichroic/emission filter sets optimised for spectral separation of compatible fluorophores as previously described (Maric et al. [36 (link)]). Antibodies were then stripped from sections with the addition of 5X NewBlot™ Nitro Stripping Buffer (Li-Cor, NE, USA) for 10 min at room temperature. Sections were then washed in PBS, epitope retrieval performed where necessary, and a subsequent round of immunostaining and imaging performed as above. This was completed over four rounds. Alignment of images from all four rounds was performed using a custom Python script [38 (link)]. We confirmed the effectiveness of stripping at removing previous antibodies in Additional file 1: Figure S5.

Sections were imaged using an automated fluorescence microscope; Zeiss Z2 Axioimager equipped with MetaSystems VSlide slide scanner (MetaSystems) running MetaFer (V 3.12.1) coupled with MetaXpress using a 20x magnification objective lens (0.9 NA). Images were stitched using MetaCyte software. Following image capture, the total section scan was viewed using VSViewer (V 1.1.106) (MetaSystems) software. The AON regions were delineated using several antibodies (Fig. 1c). Once the AON regions were identified, this process was applied to sequential sections. Cells with presumed intracellular α-syn were manually counted and marked for their location in the OFB using VSViewer (V 1.1.106) software. All cells with presumed intracellular α-syn were reimaged with a confocal microscope to confirm whether the α-syn was intracellular (Supplementary Fig. 1).
Confocal images were acquired using a FV1000 confocal microscope (Olympus, Japan) with a 40x magnification oil immersion lens (1.00 NA), 60 x magnification oil immersion lens (1.35 NA) or 100 x magnification oil immersion lens (1.40 NA) in a Z-series using a step size of 0.5 µm. Orthogonal projections with maximum intensity Z-projections were generated using ImageJ software.