Fluoromount g containing dapi

Fibroblasts were seeded at a density of 1 × 10³ cells/well on Millicell EZ SLIDE 8-well glass slide culture (EMD Millipore) coated with 100 mg/mL of quenched fluorescein-labelled gelatine (DQ™ Gelatin D-1–12,054, Molecular Probes, Eugene, OR, USA). Slides were then incubated overnight with 500 μg of 2D- or 3D-exosomes. Cells were rinsed in PBS with 0.05% Tween-20 and mounted with Fluoromount-G containing DAPI (Invitrogen). Samples were observed using a Zeiss Axio Imager M2 microscope equipped with an Axiocam HR Rev3 camera (Oberkochen, Germany). Fluorescence peptides, released by the enzymatic cleavage of DQ™ gelatine, were quantified using ImageJ 1.46 software and normalised for the number of cells per well.

For histological validation, cultivated CNS-resident cells were stained with a cell-type specific marker. For that purpose, on the day of processing, cells were washed once with 1X PBS before they were fixed with 4% paraformaldehyde for 15 min at RT. After three washing steps, cells were blocked for 1 h at RT with a solution consisting of 5% BSA, 1% host serum, and 0.2% Triton-X (Merck KGaA) in 1X PBS in order to avoid false-positive results. After blocking, the incubation with the primary cell-type-specific antibody (Table 4) was performed overnight at 4 °C in a similar blocking solution lacking Triton-X. On the next morning, the cells were washed three times with 1X PBS before being incubated with the corresponding fluorophore-conjugated secondary antibody (Table 4) in 1% BSA for 1 h at RT in the dark. After three washing steps, the cells were mounted on coverslips with 10 µL Fluoromount-G containing DAPI (Thermo Fisher Scientific) and stored at 4 °C in the dark. On the next morning, fluorescence images were acquired with a Zeiss Axio Scope.A1 (Zeiss, Göttingen, Germany) using 40-fold objectives.

iGFP-NLSm were fixed with 4% paraformaldehyde (PFA) in PBS and permeabilized with 0.1% Triton X-100 for 15 min. When indicated, to remove cytoplasmic soluble proteins and quantify the formation of TDP-43 pathology, cells were fixed in 4% PFA containing 1% Triton X-100 for 15 min at room temperature as previously described^{33 (link),34 (link)}. Briefly, after blocking, cells were incubated with the monoclonal antibody phospho-specific p409–410 antibody overnight at 4 °C. After three washes with dPBS, cells were incubated with appropriate secondary antibodies in a blocking buffer for 1 h at room temperature in the dark. Coverslips were mounted with Fluoromount G containing DAPI (Thermo Scientific).

Paraffin sections (5 µm) were deparaffinized as follows: 2 x 10 min in xylol, 2 x 3 min in absolute ethanol, 2 x 3 min in 96 % ethanol, 1 x 3 min in 70 % ethanol, 3 x 5 min in deionized water and 3 x 5 s in TBS buffer (Tris-Base (7,4 mmol/L), Tris-HCl (43,5 mmol/L), NaCl (150 mmol/L), pH = 7.5). For MC staining, the sections were incubated with Avidin-FITC (BioLegend) for 15 min in the dark. After washing (3 x 3 min with TBS) the sections were embedded with Fluoromount-G™ containing DAPI (Thermo Fisher Scientific) and dried for 24 h.

Fresh-frozen brains (N = 2 × 2 hemispheres) were sectioned on a cryostat (Cryostar NX-70) at 20 μm, mounted on Superfrost Plus slides (Fisher Scientific), and stored at −80°C until use. Slides were thawed, and RNAscope in situ hybridization was done according to the manufacturer’s protocol (#320293, ACD Inc., MN, USA). Briefly, slides were pretreated with Protease (#320842, ACD Inc.) and incubated with spCas9 probes (#519411, ACD Inc.) for 2 hours at 40°C. Sections were washed, and signal amplification was performed using the kit’s reagents. Last, sections were coverslipped in Fluoromount-G containing DAPI (Thermo Fisher Scientific). The 20× images were made on a Keyence BZ-X700 microscope (Keyence, Japan).