E800 microscope

Frozen brains were sliced serially to 40-μm sections using a cryostat (Leica, Nussloch, Germany). A series was mounted on microscope slides and used for standard cresyl violet staining. Further section series followed standard protocol for free-floating immunohistochemical staining. In brief, they were incubated with 0.3% H₂O₂ in PBS to quench endogenous peroxidase activity, blocked in PBS containing 5% normal serum and 1% BSA, and incubated with primary antibody in PBS containing 1% normal serum and 1% BSA overnight at 4°C. This was followed by incubation with the appropriate biotinylated secondary antibody (1:200 in PBS containing 1% normal serum and 1% BSA, for 1.5 h) and Vectastain ABC reagent for 1 h. Stainings were developed with 3,3’-diaminobenzidine, mounted on gelatinated microscope slides, dehydrated, and coverslipped with Entellan. Image analysis was performed by using a light microscope (Nikon E-800 microscope) and a computer-assisted image analysis system (Stereo Investigator Software; MicroBrightField Europe e.K., Magdeburg, Germany). The analyses included stereological counting of TH-positive or Nissl-stained neurons and determining the density of 15G7-positive aggregates per mm² in SNc.

Digital calipers manufactured by Kroeplin (#C11OT) were used to measure the mouse ears. Cryotome FE (Thermoscientific) was used to section the mouse ear tissue for histology. Zetasizer Nano (Malvern Instruments) and Nanodrop was used to quantify the size, charge and concentration of the QD samples. Nikon E800 microscope was used to image the histology sections and RT3 camera/spot advanced software (version 4.6) was used to acquire the images included in the supplementary data.

An observer blinded to the group assignment of the animals performed all stereological investigations using a computer-assisted image analysis system (Nikon E-800 microscope, Nikon digital camera DXM 1200; Stereo Investigator Software, MicroBrightField Europe e.K., Magdeburg, Germany). The optical fractionator method was applied for cell number estimations in the substantia nigra pars compacta (SNc) on the lesioned and non-lesioned side. Volumetric approximations of QA lesion, remaining and contralateral striatal size, as well as ventricles were performed by measuring the surface areas on six consecutives DARPP-32 stained sections on the anterior–posterior axis on the ipsi- and contralateral side. Then the volume was calculated by correcting for section thickness and sample frequency by the following formula: volume (mm³) = sum of areas (mm²) x 40 μm x 36.

The renal tissues were excised, fixed in 4% PFA and embedded in paraffin wax. Subsequently, renal tissues were sectioned into 4-μm slices. To assess glomerular matrix accumulation, slides were stained with periodic acid-Schiff (PAS). For immunohistochemical staining, slides were deparaffinized, and then blocked with 10% BAS at room temperature. Tissues were incubated overnight at 4°C with primary antibodies against WT1 (Abcam Cat# ab89901, 1:100), ZO-1 (GeneTex Cat# GTX636399, 1:100), Col1a1 (Santa Cruz Ca# sc-25,974, 1:100), β-catenin (Abcam Ca# ab32572, 1:100), and snail1 (Abcam Ca# ab53519; 1:100). The sections were then incubated with secondary antibodies for 1 h. Color development was achieved using diaminobenzidine (DAB). Histological changes in the tissue were examined using a light microscope (Nikon E800 microscope).

For immunocytochemistry, cells were fixed in 4%PFA for 10 minutes followed by 10 minutes in 0.3% triton in PBS. All antibodies except DDX3, which was made in our laboratory [31 (link)–33 (link), 37 (link), 38 (link)] is commercially available. Briefly, antibodies were diluted in 15% goat serum in DPBS (1:50 dilution) and incubated with tissue for 1 hr at room temperature. All antibodies were detected using fluorescently labeled secondary antibodies (1:200 dilution; Millipore) in 15% goat serum in DPBS for 1 hr at room temperature. Cells were counterstained with DAPI (Sigma) to detect nuclei. Negative controls were performed using secondary antibodies alone. For fluorescent quantitative analysis, images were captured using a Nikon E800 microscope and imported to Metamorph Imaging Software (Version 7.7). Total fluorescence units (FU) were calculated by dividing the number of pixels per area (3 areas measured per image), which are in arbitrary units. An adjacent area of the field that was not of interest was also measured to establish background fluorescence as previously described [44 (link)] At least, three independent experiments were performed for each treatment from which three separate 48-count tissue culture wells were counted per stain.