Coreldraw x6

Caspase activities were measured using the Caspase-Glo®8 (#G8201) and Caspase-Glo®3/7 (#G8093) assays from Promega, following the manufacturer’s instructions. Equal volumes of cell suspension, and Caspase-Glo® reagent were placed in 96-well plates and incubated for 45 min at room temperature before luminescence was monitored using a Perkin Elmer Wallac microplate reader. Cell numbers were calculated at time of harvest and used to normalize the assay results. Figures were prepared using CorelDRAW X6.

A Leica DM 1000 epifluorescence microscope (Leica, Wetzlar, Germany) was used for gross morphological analysis of 318 animals. 64 of them were selected for detailed examinations using a Leica TCS SP5 II confocal laser scanning microscope (CLSM) (Leica, Wetzlar, Germany). Optical sections between 0.2 and 0.5 μm of the whole-mounts were generated and digitally merged into maximum projections. In order to depict the 3-dimensionality of the specimens and to accentuate the regions of interests, the confocal stacks were analyzed with the 3D-reconstruction software Imaris 7.3 (Bitplane, Zürich, Switzerland). Adjustment and optimization of contrast and brightness as well as the conversion into black and white images was performed with Photoshop CS 6 (Adobe Systems, San Jose, CA, USA). Line drawings were generated with Corel Draw X 6 (Corel Corporation, Ottawa, Ontario, Canada).

Data were analyzed using IBM SPSS Statistics software and were visualized using GraphPad Prism and CorelDRAW X6 software. Pearson's chi-squared and Mann–Whitney test revealed no sex- and age-related differences between MSA patients and controls nor differences in group size and sex between MBP29-hα-syn mice and control mice. To determine normal distribution of parameters, the Shapiro–Wilk test was used. Even though most of the parameters were normally distributed, the Mann–Whitney test (non-parametric, unpaired) was chosen for all analyses due to low sample sizes. To evaluate disease-related progression in gait parameters of MBP29-hα-syn mice at 8 and 16 weeks of age, the Wilcoxon signed-rank test (non-parametric, paired) was performed. All parameters expressed in length units were normalized to the mean bodyweight of all mice at each time point to avoid confounding effects of bodyweight on gait parameters due to mixed-sex cohorts, weight gain during gait analysis, and bodyweight differences between sex, transgenic mice and controls (Additional file 1. Table S1). Unless indicated differently, all analyses are expressed as mean + standard deviation. Effect size r was calculated (according to Rosenthal 1991 [34 (link)]) using the z-score (obtained from Mann–Whitney test). P values ≤ 0.05 were considered statistically significant.

The isolation and cryopreservation of the brain-infiltrating lymphocytes (BILs) have been previously described (21 (link)). In brief, fresh brain tissue was finely minced in dissociation solution (HBSS with 20 mM HEPES pH7.0, 5 mM glucose, and 50 U/ml penicillin/streptomycin), then digested overnight at room temperature in dissociation solution containing 0.5 mg/ml Type IV collagenase (Worthington Biochemical Corp., Lakewod, NJ, USA) and 5% filtered human serum (Mediatech Inc., Manassas, VA, USA). BILs were obtained by fractionation on a 30%: 70% Percoll^® (SigmaAldrich, St. Louis, MO, USA) step gradient in RPMI containing 20 mM HEPES. BILs were stained with the following antibodies: APC-efluor^® 780-conjugated CD3 (clone UCHT1; eBioscience Inc., San Diego, CA, USA), PE/Cy7-conjugated CD4 (clone SK3; eBioscience Inc.), PerCP/Cy5.5-conjugated CD8 (clone RPA-T8; eBioscience Inc.), APC-conjugated TCR αβ (clone IP26; eBioscience Inc.), FITC-conjugated TCR γδ (clone B1.1; eBioscience Inc), and PE-conjugated CD103 (clone B-Ly7; eBioscience Inc). Data were acquired on an analytical LSRII flow cytometer (Becton Dickinson, San Jose, CA, USA), and were analyzed with FlowJo software (TreeStar Inc., Ashland, OR, USA); histograms were exported into CorelDrawX6 (Corel Corporation, Ottawa, ON, Canada). Statistical analysis and graphing utilized R-project programs (www.r-project.org).

Data were analyzed and visualized using GraphPad Prism 9.5.0 and CorelDraw X6 software. Shapiro–Wilk test was performed for determination of normal distribution of expression levels and cell counts. Welch’s t-test was conducted for determination of statistical significance. If no Gaussian distribution was determined, statistical analysis was performed using Mann–Whitney–U test. Unless otherwise stated, all graphs represent mean values and standard deviation. P values were defined as statistically significant at p < 0.05.

Data are expressed as mean ± standard deviation. Statistical analysis was performed with SPSS 21.0 for Windows (SPSS Inc., Chicago, IL, USA). Results were interpreted statistically using the non-parametric Kruskal–Wallis test and the Mann–Whitney U-Test. The level of statistical significance was set at p ≤ 0.05. All graphics were created with GraphPad 8.0. Figures were created with CorelDRAW X6. The brightness, contrast, and intensity of the depicted images were adapted for better perceptibility. Adaptations were made to the entire picture.

Microscopic images were acquired with a Zeiss AxioCam HRc digital camera (4164 × 3120 pixels; Carl Zeiss MicroImaging, Jena, Germany) attached to a Zeiss Axiophot microscope (Zeiss) and AxioVision software (version 4.8.2; Zeiss) using a 10x and 100x objective. The final figures were assembled using Corel Photo-Paint X6 and Corel Draw X6 (both versions 16.1.0.843; Corel, Ottawa, Canada). Only minor adjustments of contrast and brightness were made, without altering the appearance of the original images.