Cm1950

Animals were deeply anesthetized with isoflurane before decapitation. Brains then were rapidly removed, frozen on dry ice, and embedded in tissue freezing media (Tissue-Tek O.C.T. compound). 20 µm coronal sections were prepared on a cryostat (Leica Biosystems, CM1950) and mounted onto SuperFrost Plus glass slides (VWR) at 100 µm intervals between slices in a set. Slices were rapidly refrozen once mounted, and stored at −80°C before staining. Multiplexed fluorescent in situ hybridization was performed using the ACDBio RNAscope V1 reagents and protocols. Briefly, slices were fixed in 4% paraformaldehyde at 4°C for 15 min and dehydrated through washing steps in ethanol at increasing concentrations (50%, 70%, 100%) before protease digestion (Protease III, 10 min at room temperature). Probes and amplification/detection reagents were applied to the tissue sections and incubated under conditions stated in the V1 detection protocol provided by ACDBio. Sections were counterstained using DAPI provided in the V1 detection reagent kits and mounted in ProLong Gold mounting media (ThermoFisher Scientific P36934). Single-plane tiled images covering the DRN were scanned using a confocal microscope (Leica SP8X, 1.40 NA 63X magnification oil immersion objective). Tile merging was performing in the Leica LAS X software, with a 10% tile overlap and statistical blending.

For detection of MPs, representative post-fixed brain, lung, heart, liver, kidney, GI, and spleen samples from control and high-dose groups from both young and old cohorts were examined. Frozen embedded tissues were sectioned at 20 μm using a cryostat (Leica BioSystems, CM1950, Wetzlar, Germany) taken at −21 °C and collected onto glass slides (VWR Colorfrost^® Plus, Radnor, PA, USA). The sections were allowed to dry at 30 °C for 5–10 min before being circled using a wax pen. The sections were washed for 5 min in TBS, permeabilized with 0.3% Triton X-100 (Sigma, St. Louis, MO, USA) in TBS for 30 min, and stained with DAPI (1:5000, 5.08741.0001, MilliporeSigma, Burlington, MA, USA) for 10 min. Sections were washed again in TBS for 5 min and coverslipped (VWR, Radnor, PA, USA) with aqueous anti-fading mounting medium (20 mM Tris pH 8.0, 0.5% N-propyl gallate, 50% glycerol). Fluorescence imaging (Leica THUNDER DMi8 3D Fluorescence Imaging System, Leica Biosystems, Wetzlar, Germany and LAS X 3D Analysis Software v. 2018.7.3, Leica Biosystems, Wetzlar, Germany) was used to identify the dyed red fluorescent polystyrene particles using a TRITC (550 nm) filter.