Colorfrost plus

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.