Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

Evonuk, Kirsten S.; Moseley, Carson E.; Doyle, Ryan E.; Weaver, Casey T.; DeSilva, Tara M.

Add ABC reagent to the circled area for 30 min. 13. Flick solution off the slides and wash 3 times in 1x PBS for 5 min, then 2 times in water for 5 min. Make 3,3'-diaminobenzidine (DAB) solution (see Materials List) and add it to cover the sections. NOTE: This step requires a microscope to observe optimal detection time of staining and must be done for the same amount of time for slides to be compared. 14. Wash slides 3 times in water for 5 min each. Dehydrate the tissue by placing in the following solutions for 2 min each: 70% ethanol in water, 95% ethanol in water, 100% ethanol in water, 50% xylenes and 50% ethanol, 100% xylenes. Seal a coverslip on the slide with a resinous mounting medium. 15. Alternatively, perform immunofluorescent staining as previously described 11 to assess reactive gliosis using antibodies against Iba-1 and GFAP. 16. Take images of each spinal cord section (stained with respective antibodies using DAB) with a 4X, 0.13 NA objective and save the images as .tiff. Alternatively, take images of the corpus callosum and cingulum bundle in the left or right brain hemisphere using a 20X, 0.50 NA objective and save the images as .tiff. For a more comprehensive determination of lesion load in the brain, it is beneficial to include both hemispheres in analyses. De-noise the image by going to Process > Subtract Background and set the "Rolling ball radius" to at least the size of the largest object that is not part of the background (see the ImageJ user guide at http://rsbweb.nih.gov/ij/docs/guide/146-29.html). NOTE: For a 4X image of Iba-1 staining we use 4.0 and for GFAP we use 50.0, but these numbers may vary depending on image magnification and staining intensity. 3. Check "Sliding parabloid" and click "OK". Go to Image > Adjust > Threshold… and set the lower threshold level (the top bar) using the sliding bars. Include only staining that is cellular and be consistent across images. For images with dark backgrounds (applies to fluorescent staining only), ensure that the "Dark background" box is checked. 4. Go to Analyze > Set Measurements… and select "Area fraction" (gives the percent of thresholded area within the region of interest).