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Eli Papa 17:21, 14 January 2008 (CST)

Prepare immunogen

  • 50 ug of antigen
  • 200ul of final injection volume per mice
    • 100ul of antigen + PBS
    • 100ul of adjuvant
  • allow some extra room for dead volumes in syringes, etc (prepare for 4 mice instead of 3, 8 mice instead of 6, etc)

NOTE: use complete Freud's adjuvant only in the primary injection. Any booster should be done by incomplete adjuvant or otherwise the 2ndary response may be lethal.

  1. load antigen + pbs in an eppendorf
  2. syringe adjuvant out of bottle (gauge 21 is recommended, nothing much thinner). DO NOT MIX at this point
    • can put into eppendorf and then pipette to be more precise (allowing sufficient time for the pipette to aspirate the viscous material) or leave in the 3ml syringe.
  3. load into 3ml syringe using 21g
  4. connect syringe to 3-way (one screw), making sure the screw side is closed
  5. connect other syringe
  6. mix adjuvant + antigen by repeatedly pushing and pulling. (push with one, pull with the other)
  7. go to mouse room


  • prepare your cage
  • prepare injection
  1. push all the immunogen in one of the two 3ml syringes
  2. screw off the empty 3ml syringe
  3. loosen the 1ml syringe by moving piston up and down a couple of times
  4. plug the 1ml syringe, making sure you have a good hold and can apply pressure (there is no screw)
  5. while holding the 1ml syringe (otherwise it flies away), push immunogen into it from the 3ml syringe
  6. take the 1ml syringe off and cap with a 25g 5/8 (nothing thinner as the adjuvant does not flow much)

transfer all immunogen emulsion

  • immobilize each mice and inject IP.


  • generally, 10 days should pass after primary immunization. longer is fine, shorter (min ~ 7 days) can also work if one needs to rush. Consider that the peak cellular response occurs at days 4-7.
  • booster injections can be done as close as 1 wk. from each other