Mice: Female C57Bl/6 mice are chosen to induce the EAE model; C57BL/6 mouse displays chronic–progressive disease following active EAE induction with the MOG peptide 35–55 that resembles the later stages of MS. This chronic disease is characterized by sustained priming antigen-specific T cell responses, and it has been proved to be an important model to dissect regulatory and immunopathomechanistic pathways in EAE (14).

Experimental setup: Allocate mice to four groups (at least ten animals per group)—see Notes 6 and 7 ) and conduct DNA vaccination either with the myelin DNA vaccines (MOG, PLP, or MBP), or with the vehicle (empty pCi plasmid) as control. For prophylactic schedule, mice are injected intramuscularly at 4 and 2 weeks before EAE induction. For therapeutic treatment, mice receive intramuscular (i.m.) injections of DNA at disease onset (day 10 post EAE induction) and a second dose 2 weeks later.

Prepare 100 μg of each plasmid DNA in a volume of 100 μl endotoxin-free PBS per mouse and transfer from the vial into a 1 ml syringe using a 22–25 gauge needle.

Shave the flank of the animals to be treated with electric clippers. Clean the area with ethanol to ensure full removal of oil, dust, and dander.

Anesthetize the animals with inhaled isoflurane (5 %) in an acrylic glass nebulizing chamber and maintain light anesthesia with isoflurane (3 %) during the treatment by placing the snout into an inhalation tube.

Inject each mouse with a total volume of 100 μl of plasmid DNA (1 μg/μl), by two i.m. injections with 50 μl into tibialis anterior muscle of both hind legs with a 1 ml syringe using a 27 gauge needle.

Estimate the amount of peptide and CFA for the immunization: according to our experience using the EAE model, immunization of mice with 50 μg of the MOG_35-55 peptide in 200 μl of an emulsion with CFA is sufficient for the reproducible induction of EAE at high incidence.

Emulsion volume: Each mouse receives 200 μl of emulsion. To calculate the volume, consider in excess because there is a waste during the emulsification process (e.g., for 10 mice, instead of 2 ml prepare 2.4 ml of emulsion, calculated considering 12 mice).

Emulsion composition: Each mouse receives 50 μg of MOG_35-55 in complete Freund’s adjuvant (CFA), that is incomplete Freund’s adjuvant (IFA) supplemented with 4 mg/ml of Mycobacterium tuberculosis.

Prepare the emulsion: The solutions of MOG in PBS (0.5 mg/ml) and Mycobacterium tuberculosis in IFA (4 mg/ml) are prepared separately, in two different 2 ml tubes, and then mixed in a 1:1 volume ratio (usually we prepare emulsion for 10 mice: 2.4 ml of emulsion: 1.2 ml of 0.5 mg/ml MOG in PBS and 1.2 ml of 4 mg/ml Mycobacterium tuberculosis in IFA). Mix CFA and peptide solution by repetitive passage between two glass syringes connected by a stopcock after removing as much air from the two syringes as possible. Emulsify for 6–8 min, until the solution is firm enough (see Note 8 ).

Load plastic syringes with emulsion: Pump all of the emulsion into one of the 2 ml syringes. Remove the empty 2 ml syringe from the stopcock and replace it with a 1 ml syringe. Slowly transfer 600 μl emulsion (enough for three mice) without air, and add to the syringe a 26-gauge needle. Repeat until all of the emulsion is dispensed into plastic syringes.

Anesthetize mice with 50 μl/20 g body weight with a combination of Ketamine (37 mg/kg) and xylazine (5.5 mg/kg), via intraperitoneal injection using a 27 G syringe.