Emulsify equal volume of antigen plus Freund’s complete adjuvant or Titremax using glass syringes connected by 3-way stop cock; use anywhere from 50:g-100:g of antigen. A good emulsion is white and very thick and will not seperate. If you use Titremax, refer to the methods book since you need to use much smaller volumes.
(1ml adjuvant plus 1ml antigen--dilute antigen with saline(0.9% NaCl, found in cold room) if required.
Inject subcutaneously into back of neck, no more than 800:l in one spot. It is best to inject ~400:l into as many places as possible. Use 20 gauge needle for best results.
Record date of primary injection (make a sheet of injection schedules, name of rabbit, name of antigen, quantity of injection etc.).
FIRST BOOSTS
2 to 3 weeks after primary injection.
You may inject the same amount of antigen or half as much depending on what you have available.
Methods are the same except you will use FREUNDS INCOMPLETE ADJUVANT for all boosts.
NOTES: Each person is responsible for keeping track of dates for their own rabbits and should let Mina know when boosts/bleeds are due as she will be responsible for this aspect.
You are responsible for processing your own blood (SEE BELOW).
TEST BLEEDS
Place anticoagulant into the syringe (3.8% Na citrate use at 1:10), attach to a 23 gauge butterfly and push the anticoagulant to the end of the butterfly. 3ml of blood is adequate for a test bleed.
Insert the butterfly into the central artery of the rabbits ear. Before doing so stimulate the blood flow by gently rubbing the ear or warming it with hot water compresses.
Remove 3ml of blood into a 3cc syringe attached to the butterfly. If you need to use the central artery, stick to one ear only. You need the other ear for large bleeds.
PROCESSING BLOOD AND COAGULATING PLASMA
Spin the blood in microfuge tubes in the Hermle at 3000rpm for 10 minutes. If you have larger volumes then use the swinging bucket rotor.
Remove the plasma and place into a fresh tube.
Add 1-5 units of thrombin (1 unit should do for small volumes; use more for larger volumes).
Incubate the plasma at 37º C until it clots.
Remove fibrin with wooden applicator sticks.
Spin remaining fibrin out, in the Hermle. Freeze serum at -70ºC.
Thrombin is kept in 1u/ul aliquots in the 200X PIN box in the -70ºC freezer; reorder from Sigma
