Tissues were fixed in 4% formaldehyde overnight, dehydrated in an increasing gradient of ethanol and embedded in paraffin. In EAE experiments, parts of the vertebral column containing the spinal cord were fixed, decalcified in 0.5% EDTA for a week prior to embedding. Ten-micrometer sections were made and then dewaxed and rehydrated in an ethanol gradient. For FoxP3 and CD4 stainings, heat antigen retrieval was performed and endogenous peroxidase was quenched by a 10-min incubation in 1% H2O2. After blocking with 2% NGS, immunostaining was performed using a FoxP3 antibody (Abcam) and a secondary anti-rabbit-AP (Sigma), CD4 (Abcam) or proinsulin (Abcam) and Vectastain ABC kit according to the manufacturer's protocol. DAB solution (Sigma) was used for signal detection with HRP-conjugated antibodies or NBT/BCIP (Roche) with AP-conjugated antibodies. Proinsulin-stained sections were counterstained with hematoxylin.
Images were captured using a LMD7000 (Leica) microscope and quantified manually using ImageJ cell counter. At least three sections from each mouse separated from each other by 50 μm were analyzed.
Images were captured using a LMD7000 (Leica) microscope and quantified manually using ImageJ cell counter. At least three sections from each mouse separated from each other by 50 μm were analyzed.