Image pro analyzer software

Immunohistochemistry for Iba1 in the frontal cortex and putamen showed resident microglia and perivascular macrophages (Fig. 1f), and that for GFAP showed diffusely distributed astroglia of variable density (Fig. 1g), as previously described [58 (link)]. For quantification of Iba1 and GFAP immunoreactivity density, the DAB tissue slides were scanned using a slide scanner (20x objective, Aperio ScanScope GL, Leica Biosystems, Buffalo Grove, Illinois, USA), and a square of 3,000 × 3,000 μm² was extracted from the frontal cortex and putamen. Using the Image-Pro Analyzer software (Version 6.3, Media Cybernetics, Bethesda, Maryland, USA), the DAB intensity was quantified within each area of interest, i.e. the frontal cortical layers II-VI and putamen [59 (link)]. The DAB intensity per unit area (i.e. DAB density) was calculated [62 (link)]. To adjust for between-batch variation, one brain section from the same positive tissue control block was included in each immunostaining batch. The control DAB density value (in a specified area) was used to normalize all the DAB density values of studied cases in the same batch, giving rise to the immunoreactivity density values (continuous). Finally, each of the Iba1 and GFAP immunoreactivity density values was graded as mild (≤ the median) or marked (> the median) gliosis (categorical).

Following treatment with PMO, equal numbers of live RA FLS were resuspended in FLS medium containing 5% FBS and allowed to adhere onto coverslips coated with 20 µg/ml fibronectin (FN) at 37°C for 15, 30 and 60 min. Cells were fixed in 4% para-formaldehyde for 5 min, permeabilized in 0.2% Triton X-100 for 2 min, and stained with 5 U/ml Alexa Fluor® 568 (AF 568)-conjugated phalloidin and 2 µg/ml Hoechst for 20 min (Life Technologies). Samples were imaged with an Olympus FV10i Laser Scanning Confocal Microscope (Olympus, Center Valley, PA). Using the FV10i acquisition software, each coverslip was separated into four nine-paneled mega-images. Each panel (1024×1024) was acquired with a 10× objective and then stitched together, through a 10% overlap, with the Olympus FluoView 1000 imaging software. Total cell number and cell areas for each panel were calculated using Image Pro Analyzer software (Media Cybernetics, Rockville, MD).