Microsuite 5 software

Immunofluorescence analysis was performed as described earlier (47 (link)–49 (link)). Briefly, coverslips containing 100 to 200 cells/mm² were fixed with 4% paraformaldehyde followed by treatment with cold ethanol and two rinses in PBS. Samples were blocked with 3% bovine serum albumin (BSA) in PBS–Tween 20 (PBST) for 30 min and incubated in PBST containing 1% BSA and anti–IL-11 or anti- CD3. After three washes in PBST (15 min each), slides were further incubated with Cy2 (Jackson ImmunoResearch Laboratories Inc.). For negative controls, a set of culture slides was incubated under similar conditions without the primary antibodies. The samples were mounted and observed under an Olympus IX81 fluorescence microscope. Counting analysis was performed using Olympus MicroSuite V software with the help of a touch counting module.

After 8 weeks of treatment, mice were sacrificed and their brains fixed, embedded, and processed. Sections (30 μm) were made from different brain regions (motor cortex and striatum) using a Leica Cryostat and immunofluorescence staining on fresh frozen sections was performed as described (Corbett et al. 2015 (link), Roy et al. 2015 (link), Roy et al. 2016 (link)). Briefly, before adding blocking buffer (2% BSA in PBS), sections were incubated in 100 mM glycine for 20 min for reducing autofluorescence. For details on antibody concentrations, please see Table 1. The samples were mounted and observed under Olympus IX41 fluorescence microscope. Counting analysis was performed using the Olympus Microsuite V software for imaging applications with the help of touch counting module (Corbett et al. 2015 (link), Roy et al. 2015 (link)). After acquiring images under 20 X objective lens, images were further analyzed as follows. Before counting cells, the entire image area was calibrated with the help of a rectangular box available in the touch counting panel. Once the area of the image was measured, touch counting program was applied to count number of fluorescent signals using simple mouse click method. Next, the total number of signals in a given area was divided by the total area of the image and presented as number of cells per square millimeter unit.

It was performed by monitoring subunit c of mitochondrial ATP synthase (SCMAS) by immunofluorescence. Please see Table 1 for details on antibody dilutions. DAPI was used to monitor nucleus. SCMAS-associated fluorescence intensity was quantified by using the Olympus Microsuite V software. Briefly, captured images were opened in the infinity image viewer and the contour was drawn around the granules to obtain the fluorescence intensity.