Before you start
- Approval: this procedure must be covered by your institution’s approved animal protocol, including humane endpoints.
- Use age- and sex-matched mice (commonly 8–12-week-old females).
- Prepare 20–30% extra emulsion to cover syringe losses.
Reagents and materials
Totals are for 10 mice (+10% extra). Per-mouse amounts are in the middle column.
| Reagent | Per mouse | Total |
|---|---|---|
| MOG₃₅₋₅₅ peptide (100–200 µg per mouse)dissolved in PBS at 2 mg/mL for a 200 µg dose | 200 µg | 200 µg |
| PBS for peptide solution | 100 µL | 100 µL |
| CFA with M. tuberculosis H37Ra (e.g. 4 mg/mL) | 100 µL | 100 µL |
| Pertussis toxin (100–300 ng per injection, two injections)common dose 200 ng × 2 | 400 ng | 400 ng |
| PBS for pertussis toxin injections | 0.4 mL | 0.4 mL |
Equipment
- Two glass Luer-lock syringes and a three-way stopcock or emulsifying needle
- 1 mL syringes with 25–27 G needles
- Scale for body weight
Procedure
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Prepare the emulsion
Mix equal volumes of peptide-in-PBS and CFA and emulsify between two Luer-lock syringes (or by sonication) until thick and white, typically 10 min or more on ice.
Test the emulsion
Drop a little onto cold water: a stable emulsion stays as a drop and does not spread.
Immunise (day 0)
Inject 200 µL emulsion subcutaneously, divided between two sites on the flanks (or as your protocol states).
Pertussis toxin (day 0)
Inject pertussis toxin intraperitoneally in 100–200 µL PBS on the same day.
Pertussis toxin (day 2)
Repeat the pertussis toxin injection 48 h later (some protocols use day 1).
Weigh and score daily
From about day 7, weigh mice and score clinical signs every day at the same time, ideally blinded to group.
Supportive care
From score ≥ 2, place wet food and gel on the cage floor and check hydration. Apply humane endpoints as approved.
Critical points
- A common clinical scale: 0 no signs; 1 limp tail; 2 limp tail and hind-limb weakness; 3 partial hind-limb paralysis; 3.5 complete hind-limb paralysis; 4 hind-limb paralysis with forelimb weakness; 5 moribund. Half points are used between grades. Use the scale and endpoints in your approved protocol.
- Onset is usually around days 9–14 with peak around days 14–18; incidence and severity depend on peptide, adjuvant and pertussis toxin lots, sex, age and housing.
- Emulsion quality is the most common cause of failed induction.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Low incidence | Check emulsion stability, pertussis toxin activity (lot, storage, dilution), mouse age/sex, and stress in the facility. |
| Very severe early disease | Reduce pertussis toxin dose; check dosing calculations. |
| Variable scoring | Train scorers together and score blinded. |
Safety
CFA can cause severe reactions in people: avoid self-injection and eye contact; use a sharps container. Pertussis toxin is a potent toxin: handle with gloves and follow your institution’s rules.
Further reading
- Stromnes IM, Goverman JM. Active induction of experimental allergic encephalomyelitis. Nature Protocols (2006).
- Bittner S et al. Myelin oligodendrocyte glycoprotein (MOG35-55) induced EAE in C57BL/6 mice. J Vis Exp (2014).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.