Encyclopedia of Experiments: Immunology
É necessário ter uma assinatura JoVE para assistir este Faça login ou comece sua avaliação gratuita.
Transcript
To prepare the immunization mixture, connect two 10-milliliter Luer-lock lock tip glass syringes to the short arms of a three-way stopcock, and close the third outlet with the long arm. Next, pipette 1 milliliter of IFA and 50 micrograms rMOG together, and adjust the mixture to a final volume of 2 milliliters with sterile PBS.
Place the diluted IFA and rMOG mixture in the open syringe. Then, insert the piston gently whilst maintaining a loose pressure on the opposite piston. Emulsify the inoculum by driving it from one syringe to the other via pushing the pistons back and forth until it is white and viscous.
Next, fix a 1-milliliter Luer-lock syringe to the open short arm of the three-way stopcock and fill it with inoculum. Distribute all inoculum to 1-milliliter syringes, and keep them on ice until the injection. Subsequently, inject 200 microliters of the IFA and rMOG mixture subcutaneously at the tail base under temporary isoflurane anesthesia, using a 21-gauge needle.
For intracerebral cytokine injection, adjust the length of the connector cannula to 2 millimeters. Fill a 1-milliliter syringe with the cytokine mixture. Then, connect the syringe to the connector cannula.
Next, fill the cannula with the cytokine mixture, avoid creating any bubbles. Then, mount the syringe onto the programmable syringe pump and program it to inject at a rate of 0.2 microliters per minute. Start the pump and keep it working in order to avoid an air bubble formation at the tip of the cannula.
Afterward, remove the catheter cap with the inlet. Insert the connector cannula into the catheter, screw, and tighten it. Then, allow the injection to proceed for 10 minutes before stopping the pump.
Leave the cannula inside the catheter for 20 minutes to allow the injected volume to fully diffuse. Subsequently, unscrew the connector cannula and remove it slowly to avoid a vacuum effect. Reattach the catheter cap with the inlet and screw it. Allow the animal to recover from the anesthesia in a cage.
