Nis elements version 3

Yeast cells collected by centrifugation (4000 g, 5 min,) and suspended in 200 μL phosphate buffered saline were stained with 10 μg/mL concanavalin A–Alexa Fluor 594 conjugate (Molecular Probes, Eugene, OR) and 20 μg/mL Fluorescent Brightener 28 (Sigma) for 25 min in the dark. Then, cells were washed twice in PBS and fixed with 4% paraformaldehyde for 30 min. Fluorescence-stained sections were examined under a Nikon A1 RSI microscope with a magnification of ×60 at constant Z-steps of 1 μm. The laser confocal system comprised a 65-mW multi-Ar laser. Three-dimensional (3D) images were processed with NIS elements version 3.21 (Nikon Instruments Inc.) and Volocity 3D image analysis software version 6.01 (PerkinElmer). Cell size measurements and total cell volume were determined using ImageJ software.

Human immune cells were incubated with GFP-tagged C. albicans under the same coculture conditions as described above. Cells were incubated in Lab-Tek slides for 6 days at 37°C and 5% CO₂. After being washed twice in PBS and fixed with 4% paraformaldehyde for 30 min, cells were permeabilized with 100% acetone. Nonspecific binding sites were blocked with 1% bovine serum albumin (BSA) in PBS for 30 min. Rhodamine-conjugated phalloidin (Wako; Osaka, Japan) was added at a 1:600 dilution to stain actin filaments for 30 min at room temperature. Nuclear DNA was stained with Hoechst in PBS for 1 min. Slides were air dried and mounted with Vectashield medium. Fluorescence-stained sections were examined under a Nikon A1 RSI microscope with a magnification of ×20 at constant Z-steps of 1 μm. The laser confocal system comprised a 65-mW multi-Ar laser. Three-dimensional (3D) images were processed with NIS elements version 3.21 (Nikon Instruments Inc.) and Volocity 3D image analysis software version 6.01 (PerkinElmer).

Once a WML was identified on an LFB section, the WML area was roughly delineated by hand using a permanent marker and was used to identify the same corresponding WML area in the adjacent Bielschowsky stained section. Within the WML area and NAWM on the Bielschowsky section, five randomly selected areas had 3 × 3 single images at 200 × magnification which were captured and using NIS elements version 3.0 (Nikon, Surrey, UK). Individual and standardized Red Green Blue (RGB) thresholds for Bielschowsky’s staining were applied until all visible axons were included with necessary manual setting of regions of interest to exclude vessels and perivascular spaces and artifacts. The area covered by Bielschowsky’s stain was measured and its percentage of the total measured area is calculated and expressed as Bielschowsky’s area (BiA), and mean regional values were calculated for the WML area and NAWM per case.

Retinal sections and cell preparations were viewed using a Leica DM LB2 microscope with Nikon Digital Sight DS-U2 camera, using 10x, 40x and 100x objectives. Images were taken using the software NIS-Elements version 3.0, Nikon, Japan. Immunofluorescence on retinal sections was performed on at least three mice of each group, at each time-point. Immunofluorescence on cell preparations was also performed in triplicate. Confocal micrographs were taken using an Olympus Fluoview FV1000 laser scanning confocal microscope, using 20x and 60x objectives. Images were taken using the software Olympus Fluoview Ver 4.1a and are represented as single slices in the XY plane or maximum intensity projections from acquisition of z-stacks. Identical microscope settings were used when visualizing specific markers across all timepoints and treatments.