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.

2. Staining for reactive gliosis and myelin 1. When ready for staining, choose one slide each for brain and spinal cord per stain for every animal in the same (or as similar as possible) region. For the brain, choose slides showing the corpus callosum and cingulum bundle. 2. Place slides with tissue on a heat block at 70 o C for 7 min. After 7 min turn off the heat block and let the slides cool on the heat block for another 10 -15 min. This will prevent tissue sections from falling off the slides during the staining procedure. 3. Wash slides 3 times each in 1x PBS with 0.1% nonionic detergent (for intracellular antigens) or 1x PBS (for antibodies targeting surface antigens) for 5 min. NOTE: Since the antibodies used in this protocol are intracellular, nonionic detergents will be used in subsequent steps. Never allow slides to dry completely after this step. 4. Place slides in a container and cover with citrate buffer pH 3.0. To make citrate buffer, add 0.192 g anhydrous citric acid to a final volume of 100 ml in water. Adjust pH with acetic acid if above pH 3.0 or NaOH if below. 5. Incubate slides at 37 o C for 30 min and wash 3 times in 1x PBS with 0.1% nonionic detergent for 5 min. 6. Circle an area around the tissue with a hydrophobic barrier pen and place the slides in a humidified chamber (for example, a slide box containing wet paper towels). Add blocking buffer to the tissue. Incubate for 30 min at RT. NOTE: Blocking buffer consists of 1x PBS plus 0.3% nonionic detergent and the appropriate serum (5%) based on the host of the secondary antibody, i.e., horse serum for myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP), and goat serum for Iba1. 7. Flick blocking buffer off the slides and add primary antibody (1:1,000 or 0.2 µg/ml goat anti-myelin basic protein for oligodendrocytes, 1:1,000 or 1 µg/ml to 3 µg/ml mouse anti-GFAP for astrocytes, or 1:750 or 0.67 μg/ml rabbit anti-Iba1 for microglia) diluted in the appropriate blocking buffer (see step 4.2.6) to the circled area. Leave at 4 o C overnight in the humidified chamber. 8. Flick antibody in blocking buffer off the slides and wash the slides 3 times in 1x PBS with 0.1% nonionic detergent for 5 min. 9. Add secondary antibody (1:200 or 7.5 µg/ml biotinylated horse anti-mouse for MBP and GFAP, or biotinylated goat anti-rabbit for Iba1) diluted in the appropriate blocking buffer (see step 4.2.6) to the circled area and leave slides to incubate in the humidified chamber for 1 hr at RT. 10 . Flick antibody in blocking buffer off the slides and wash the slides 3 times in 1x PBS with 0.1% nonionic detergent for 5 min. 11. Prepare avidin-biotin-peroxidase complex (ABC) in immunoperoxidase (see materials list) 30 min before use and agitate on a shaker until needed in 4.2.12. Add 0.3% H 2 O 2 in methanol to the circled area for 10 min to quench endogenous peroxidase activity. 12. Flick solution off the slides and wash in 2 times in 1x PBS or 1x PBS with 0.1% nonionic detergent for 5 min, then 1 time in 1x PBS.