GeoMx spatial transcriptomics

Spatial transcriptomics was performed using the nanoString GeoMx® Digital Spatial Profiler. Sections were cut at 6 μm thickness and mounted on plus-charged slides (Epredia Colormark Plus CM-4951WPLUS-001). Slides were baked at 60 °C for 1 hour and stored at 4 °C in a vacuum sealed container containing desiccant for up to two weeks. All subsequent steps were performed using RNase-free conditions and DEPC treated water. Slides were de-paraffinized with 3 sequential 5-minute washes in xylenes, followed by 2 washes in 100% ethanol for 5 minutes, 1 wash in 95% ethanol, and 1 wash in 1x PBS. Target retrieval was performed in target retrieval reagent (10x Invitrogen 00-4956-58 EDTA pH 9.0) diluted to 1x in the BioGenex EZ-Retriever System for 10 minutes at 95 °C. Slides were then washed with 1x PBS for 5 minutes. Slides were then incubated in 0.1 μg/mL proteinase K (Invitrogen 25530-049) for 10 minutes at 37 °C and washed in 1x PBS for 5 minutes at room temperature. Slides were post fixed for 5 minutes in 10% neutral buffered formalin followed by two washes in NBF stop buffer (24.5 g Tris base and 15 g Glycine in 2 L DEPC water) for 5 minutes each and one wash in 1x PBS for 5 minutes. Slides were then incubated with hybridization probes (nanoString Cat# 121401103) diluted in Buffer R (provided in the GeoMx RNA Slide Prep FFPE- PCLN kit, catalog # 121300313) in a hybridization oven at 37 °C for 16-20 hours.

Following probe incubation, slides were washed with stringent washes (equal parts formamide and 4x SSC buffer) at 37 °C twice for 25 minutes each. Then slides were washed twice in 2x SSC buffer. Slides were incubated in 200 μL buffer W (provided in the GeoMx RNA Slide Prep FFPE- PCLN kit, catalog # 121300313) for 30 minutes and incubated in morphology markers (GFAP-488, Thermo 53-9892-82, RRID:AB_10598515, 1:400; pS129 α-synuclein (81A), BioLegend 825701, RRID:AB_256489, 1:1000; NeuN, Millipore ABN78, RRID:AB_10807945, 1:1000) at 4 °C overnight. Slides were washed 4 times in 2x SSC buffer for 3 minutes each wash. Slides were then incubated with secondary antibodies (GαRb 647, Thermo Scientific A21244, RRID:AB_2535812, 1:1000; GαIgG2a 594, Thermo Scientific A21135, RRID:AB_2535774, 1:1000) and nuclei marker Syto83 (Thermo Scientific S11364, 1:1000) in Buffer W for 1 hour at room temperature in a humidified chamber. Slides were washed 4 times in 2x SSC buffer for 3 minutes each and placed in the nanoString GeoMx® DSP instrument.

Syto83 immunofluorescence was utilized for autofocus of GeoMx imaging. Immunofluorescence for GFAP was used in identification of morphological markers to aid in fitting to the Allen Brain Institute’s mouse brain atlas. NeuN and pSyn were used for segmentation. Prior to region-of-interest (ROI) generation, the slide image was exported, and individual brains were registered to the Allen Brain Atlas CCFv3 (10.1016/j.cell.2020.04.007) using the mouse brain registration protocol. Images of registered brains were imported onto the DSP instrument and fit exactly to the slide image to enable accurate anatomical selection of ROIs. ROIs were generated using the polygon tool which aligned with cortical layer 5 or layer 6 of the anterior cingulate area (ACAd or ACAv), primary motor cortex (MOp), and secondary motor cortex (MOs). Each ROI was segmented into two areas-of-illumination (AOI): “pSyn + ” neurons positive for pS129 α-synuclein and NeuN or “pSyn-” neurons positive for NeuN and negative for pS129 α-synuclein. Probe identities in each segment were captured via UV illumination and movement to a 96-well plate.