The overall goal of this procedure is to induce an animal model of experimental autoimmune encephalomyelitis that imitates the clinical and pathophysiological features of multiple sclerosis. This is accomplished by first preparing the antigen and adjuvant and the pertussis toxin. The second step is to inject the antigen and CFA emulsion subcutaneously and the pertussis toxin intraperitoneal.
Two days later, the m are again injected with pertussis toxin intraperitoneal. Following this, the animals are weighed and their clinical score is evaluated daily. Ultimately, clinical signs of experimental autoimmune and encephalomyelitis can usually be observed between day nine and 14 post immunization.
The main advantage of this technique of existing protocols, which involve, for example, toxic agents such as rizone or different variants of EIE, is that why they all mimic features of MS that differ tremendously in underlying pathological features, such as the involvement of the adaptive immune system. Generally, individuals new to this method will struggle because of the difficult handling of the immunization. It's absolutely necessary to reduce stress for the animals and to ensure optimal immunization After its development.
This model paved the way for researchers in the field of neuroimmunology to characterize knockout animals. To identify new molecular drug targets or to test promising drug candidates To prepare the peptide solution begin by diluting lyophilized MOG 35 to 55 peptide in double distilled water to a final concentration of two milligrams per milliliter. 100 microliters of this solution is needed per mouse and some volume is lost during preparation.
So make 1.5 to two times the amount of solution needed. Store the peptide solution at minus 20 degrees Celsius to make complete frons adjuvant or CFA place 100 milligrams of desiccated mycobacterium to tuberculosis. H 37 RA into a mortar and grind it with a pestle to obtain a thin powder.
Then add 10 milliliters of incomplete frons adjuvant to obtain a 10 milligram per milliliter CFA sock solution. The CFA stock can be stored at four degrees Celsius until needed did on the day of immunization. Dilute the CFA stock solution with incomplete fres adjuvant to a final concentration of two milligrams per milliliter mixed thoroughly to resuspend the particulate material and place it on ice.
After defrosting an aliquot of the MOG 35 to 55 peptide solution. Place it on ice. Use a two milliliter syringe and a 27 gauge cannula to draw up one milliliter of the CFA.
Then use a second two milliliter syringe and a 20 gauge cannula to draw up one milliliter of the MOG 35 to 55 solution. Remove any air bubbles and connect both syringes with a three-way valve. With the three-way valve almost closed, push the emulsion from one syringe to the other and back mixing thoroughly for at least 10 minutes.
When finished, the solution should be white, stiff and viscous with no separation of phases. Now let the emulsion sit for at least 30 minutes or up to several days prior to immunization Check to be sure it is still emulsified. On the day of immunization, draw the solution into one of two syringes and connect a 27 gauge cannula.
Reconstitute 50 micrograms of pertussis toxin in 500 microliters of double distilled water. For a 100 microgram per milliliter stock solution, store the stock solution at four degrees Celsius until needed. On the immunization day, dilute the pertussis stock solution one to 50.
With PBS, prepare a syringe with 200 microliters of the diluted pertussis solution for injection, which contains 400 nanograms of pertussis toxin and draw up an additional 100 microliters for the dead space of the needle hub. After anesthetizing a mouse with iso fluorine, assess the level of anesthesia using a toe pinch. Inject 100 microliters of the antigen CFA emulsion subcutaneously into two different sites on each hind flank.
After the injection, a bulbous mass should form under the skin, which should persist throughout the experiment. Next, inject the prepared syringe containing 200 microliters of pertussis toxin intraperitoneal. Ensure that individual mice can easily be identified for daily evaluation.
For example, by color markings on the tail base.
