Dakocytomation fluorescent mounting medium

Cells were fixed with 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd., Saitama, Japan) for 10 min at 24°C, and reacted with 0.1% TritonX-100 (Sigma-Aldrich) and 5% of goat normal serum (Agilent Technologies) in PBS (Wako Pure Chemical Industries, Ltd.) for 10 min. Cells were then incubated overnight with each primary antibody (S6 Table) in PBS at 4°C. They were then incubated at 24°C with the secondary antibody for each primary antibody conjugated with Alexa Fluorescent dye (1:300 dilution, Thermo Fisher Scientific Inc.). The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (Wako Pure Chemical Industries, Ltd.) for 45 min. To prevent fading, cells were then mounted in DakoCytomation Fluorescent Mounting Medium (Agilent Technologies). Samples were observed and images were captured with an Olympus IX71 inverted microscope (Tokyo, Japan) or a Keyence BZ-X700 digital microscope (Osaka, Japan). F-actin was discerned by staining with CytoPainter Phalloidin-iFluor 594 Reagent (ab176757, Abcam).

Detached skin fibroblasts were counted (MOXI Z Mini Automated Cell Counter; ORFLO Technologies, Ketchum, ID) and seeded on three autoclaved coverslips per subject at 5.0 × 10⁴ cells per slip using a 24 well-plate. Cells were incubated at 37°C overnight before being fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) at 37°C for 15 min. Coverslips were quenched with NH₄Cl for 15 min and washed with PBS to minimize autofluorescence. Cells were permeabilized with 0.5 mL 0.2% Triton X-100 (Sigma; Oakville, ON) in PBS for 15 min, washed with PBS, and then blocked with 10% FBS (Life Technologies) in PBS for 30 min. Cover slips were simultaneously incubated with primary antibodies for the outer mitochondrial membrane protein TOMM20 (FL-145, Santa Cruz; Dallas, TX) and DNA (CBL-186, EMD Millipore; Etobicoke, ON) diluted to 1:1,000 in 5% FBS, 95% PBS (Life Technologies) at 37°C for 1 h. Cells were washed 3 x with PBS and then incubated with secondary antibodies (1:5,000) at RT for 1 h. The following secondary antibodies conjugated to fluorescent dyes were used: Alexa Fluor 488 goat anti-mouse IgG (TOMM20, Molecular Probes, Eugene, OR) and Alexa Fluor 647 goat anti-rabbit IgG (DNA, Molecular Probes). Cells were mounted with DakoCytomation fluorescent mounting medium (Agilent Technologies; Santa Clara, CA) and stored at 4°C until microscopic examination.

The study of nuclear morphologic changes induced by AD0157 was assessed by Hoechst 33258 stainings (García-Caballero et al., 2011 (link)). Thus, 5 × 10⁵ human myeloid leukemia cells/well were seeded in complete medium on 8-well Falcon humidified chamber slides and incubated at 37°C in a humidified 5% CO₂ atmosphere, with or without the indicated concentrations of AD0157 for 14 h. After incubation, cells were washed with PBS, fixed with formalin solution and stained with Hoechst 33258 (1 μg/mL in PBS), using a cytospin. Cells were mounted on slides using DAKO Cytomation Fluorescent Mounting Medium (DAKO, Denmark) and observed under a fluorescence microscope (Leica, TCS-NT, Heidelberg, Germany).

Cells fixed with 4% paraformaldehyde for 30 min at 4 °C were blocked with 3% NGS (Atom) and 0.1% (vol/vol) Triton X-100 (Sigma-Aldrich) in PBS solution. Hoechst 33342 (1:10,000, Thermo Fisher Scientific) was used for nuclei counterstain for 1 h at RT in 2% (vol/vol) NGS. Cells were coverslipped using Dako Cytomation Fluorescent Mounting Medium (Dako) and images acquired using standard filter sets either with an Olympus FSX100 microscope with a DP72 incorporated camera and FSX-BSW visualization software (Olympus, Germany) or with an Olympus FluoView™ FV1000 confocal microscope and FV10-ASW 4.2 visualization software.

Cells were cover slipped using Dako Cytomation Fluorescent Mounting Medium (Dako, Sant Just Desvern, Spain). Immunofluorescence images were acquired using standard filter sets using an Olympus FluoView TM FV1000 confocal microscope and the FV10_ASW 4.2 visualization software.
α-SYN fluorescence intensity for each condition was analyzed in an average of 150 cells from 6 different random fields at an objective magnification of 40×. Quantification of Thioflavin S positive aggregates in each condition was analyzed in an average of 20 cells from 3 different random fields by measuring the fluorescent area per cell. All quantification analyses were done using ImageJ software (NIH, USA).