Microsuite software

Age-synchronized worms were collected and washed with M9 buffer. Worms were then fixed in 2x MRWB buffer (160 mM KCl, 40 mM NaCl, 14 mM EGTA, 1 mM spermidine-HCl, 0.4 mM spermine, 30 mM PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid); pH 7.4], 0.2% ß-mercaptoethanol) with 2% paraformaldehyde at room temperature for one hour. The worms washed with M9 buffer, then dehydrated in 60% isopropanol for 15 min at room temperature and stained with 60% Oil Red O solution at room temperature overnight. Animals were mounted on 2% agarose pads and imaged at 10x magnification using an OLYMPUS BX63 with DIC using Olympus Microsuite software. Oil red O intensities were determined using ImageJ software (NIH).

A wound healing assay was performed to assess the migration ability of the cell lines. The cells were cultured to 100% confluence, in a six-well cell culture plate. A scratch was created with a 10 μL pipette tip. The culture was washed with PBS to remove cell debris, and incubated in a medium without FBS, at 37 °C. The migration distances were recorded at 0, 6, 12, 18, and 24 h. For IL-6-stimulated migration, cells were starved overnight, and wounds were created in the same manner as that mentioned above. The cells were re-fed with serum-free media and treated with 50 ng/mL of IL-6 for 24 h, or were left untreated. Images of the wounds were captured at different time points. The Olympus Microsuite software (Tokyo, Japan) was used to estimate the cell-free area of the wounds (40×). All assays were performed in triplicate.

Chondrocyte extracellular matrix formation was assessed by means of IHC analysis of type II collagen in rabbit bone sections (II-II6B3; Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, Iowa) on a Leica Bond Max (Leica Microsystems) with use of an open polymer detection system (Leica Microsystems) (Bries et al., 2012 ). Briefly, prepared sections were treated with 0.1% pronase for 20 min at 37 °C, blocked with Background Buster (INNOVEX, Richmond, CA) for 1 h and subsequently exposed and bound for 1 h to the primary antibody to type II collagen (3 μg/ml). Detection of type II collagen was achieved by means of a polymer link reaction with 3,3′-diaminobenzidine (MaxTag DAB tablets, Rockland, Limerick, PA) for 5 min followed by hematoxylin counterstaining (Bries et al., 2012 ). Appropriate negative control slides without primary antibody accompanied positive slides during each Bond Max program. Stained histological or immunohistochemical sections were examined and photographed with use of an Olympus IX-70 light microscope and MicroSuite software (Olympus America, Melville, NY) (Bries et al., 2012 ; Tank et al., 2013 (link)).

Counting of α-syn puncta was performed after reconstructing individual α-syn-ir DA cell body in surfer 12 contouring software manufactured by Golden Software, Colorado followed by counting individual puncta in touch counting module of Olympus Microsuite software. Co-localization of TH and BPOZ-2 in nigra was performed using NIH FIJI Co-localization tool. Quantitative data was presented as the mean ± SEM. Statistical significance was accessed via an unpaired two-tailed Student’s t test or an ANOVA test with Student-Newman-Keuls post hoc analysis.