Hibernate a

Mouse spinal cords were obtained at the peak of the 2nd attack, 5 days after onset of treatment. Spinal cords were mechanically disrupted in Hibernate A (Life Technologies, Grand Island, NY) using a stainless steel sieve, which was followed by enzymatic dissociation in 1 mL of Trypsin LE (Life Technologies) for 30 min at 37 °C. Cells were washed and resuspended in 10 mL of Percoll of ρ = 1.03 which was underlain in Percoll of ρ = 1.095 and centrifuged for 30 min at 500μg and 4 °C with slow acceleration and no brake. The top layer containing myelin was discarded, and leukocytes were collected from the interface between ρ = 1.03 and ρ = 1.095. After washing, cells were treated with Fc block (BD Bioscience, San Jose, CA), stained with the indicated anti-mouse antibodies [anti-CD45-v500 and 7-AAD (obtained from BD Bioscience), anti-CD3e-FITC, anti-CD4-PE, anti-CD19-efluor450 and anti-CD11b-APC (all obtained from eBioscience, San Diego, CA)] for 30 min at 4 °C, and analyzed on a BD FACS Canto II flow cytometer. Spinal cord-derived cells were stained and analyzed by pooling two to three mouse spinal cord cells per tube. Gating was determined by fluorescence-minus-one (FMO) with isotype-matched immunoglobulin control. Flow cytometry data analysis was performed using FlowJo software (Tree Star, Inc., Ashland, OR).

The MnPO was punched out of a brain slice and incubated in Hibernate A (Invitrogen, Temecula, CA) and papain (Worthington, Lakewood, NJ) (1 mg/ml) for 10 min at room temperature. After washing out the enzyme with Hibernate-A the cells were dissociated by gentle trituration with a fire-polished Pasteur pipette. The cell suspension was pelleted (1000 g for 2 min) and resuspended in Neurobasal medium (supplemented with 2% B27, 0.5 mM Glutamax and 10 mg/ml gentamycin) and then plated on poly-D-lysine/laminin coated coverslips (Biocoat, BD Biosciences, Bedford, MA). Cells were kept in culture for 5–10 days before being used for experiments.

All experiments were carried out in accordance with the guidelines for the welfare of experimental animals issued by the U.S. Public Health Service Policy on Human Care and Use of Laboratory Animals and the University of California-Los Angeles (UCLA) Animal Research Committee. Adult Sprague-Dawley rats (100–300 g, Charles River, Wilmington, MA, RRID:RGD_734476), and wild-type C57BL/6 mice (20–30 g; Jackson Laboratory, Bar Harbor, ME, RRID:IMSR_JAX:000664) of both sexes were used for these studies. Animals were 2-3 months old at the time of the experiments.
Animals were deeply anesthetized with 1–3% isoflurane (Abbott Laboratories, North Chicago, IL), and killed by decapitation or cervical dislocation. The eyes were removed and dissected in Hibernate A (Invitrogen, Carlsbad, CA). For vertical cryosections of the retina, the eyecups were fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), pH 7.4, for 15–60 minutes at room temperature (RT). Eyecups were then transferred to 20% sucrose in PB for an hour or 30% sucrose in PB overnight at 4°C. The eyecups were embedded in optimal cutting temperature medium (Sakura Finetek Inc., Torrance, CA) and sectioned at 12–14 μm using a Leica CM3050S or Leica CM 1900 cryostat (Leica Microsystems, Buffalo Grove, IL) and tissue sections were mounted onto gelatin-coated slides. Sections were stored at −20°C until immunostaining.