Standard Operating Procedure

 

Epicutaneous immunization of mice

 

Hazards:

CpG ODN1826 is an immunological adjuvant.  Toxilogical data are not available.  Handle as potentially harmful: May cause eye irritation. May cause skin irritation.  May be harmful if absorbed through the skin. May be harmful if inhaled, May be harmful if swallowed

IMIQUIMOD is an immunological adjuvant.  Toxilogical data are not available.  Handle as potentially harmful: May cause eye irritation. May cause skin irritation.  May be harmful if absorbed through the skin. May be harmful if inhaled, May be harmful if swallowed

Ketamine and Buprenorphine are controlled substances and anyone working with these needs to take the training session and be registered the SBOP (State Board of Pharmacy).  Go to this website to register for the training https://onestop2.umn.edu/training/courseDetail.jsp?course=RA0001

EXPOSURE TO ANIMALS: Personnel involved with animals may be exposed to bites, scratches, infectious agents, and allergens.

TETANOUS: a rare but potentially fatal bacterial infection is possible after any animal bite.

See also the Boynton Health Service brochures on animal bites and allergies in this manual.

CARBON DIOXIDE: toxic gas, avoid inhaling.

SHARPS: take appropriate cautions when working with needles

 

Protection:

Wear gloves and lab coat.

Wear a face mask if youÕre allergic to mice or to prevent the onset of allergies.

Have current tetanus immunization.

If biten by a mouse, immediately wash wound with soap and water. Seek medical assistance from Boynton Health Service, if necessary (625-7900).

 

 

Waste:

Animal carcasses must be frozen and disposed of by Research Animal Resources. There is a freezer on the 2nd floor of Nils Hasselmo Hall.

Needles must be disposed of in a biohazard sharps container.

 

Spill clean-up: Isolate the area. Use appropriate tools to put materials into a convenient waste container. 

 

Procedure:

 

Prepare reagents:

-antigen/adjuvant: volume typically 50ul per mouse

-Ketamine xylazine mix: load 120ul/mouse into a 1cc syringe

-Buprenorphine: dilute stock 1:10 in PBS, load 30ul/mouse into a 0.3 cc syringe

-Eye moisture salve, electric shaver

 

1. Anesthesia: Inject animals with 80ul ketamine/xylazine mix i.p. (use 1 cc tuberculin syringe, with 28g needle)

            Note: as this anesthetic is a controlled substance, the lab stock is kept in a locked box under JieÕs bench.  Code (now) is 1-1-2-4.  From commercial stocks of 100 mg/ml Ketmine and 100 mg/ml xylazine, technicians made stocks that are 18 mg/ml ketamine and 1.8 mg/ml xylazine (10ml ketamine + 1ml xylazine into 44ml PBS).  These are in 15 ml conical tubes.  The precise amount used should be recorded in the Disposition Log in the Controlled substances notebook above JieÕs desk.

            We are approved to inject 100ul of this i.p., which is 90 mg/kg ketamine for the average mouse (25 g).  Use 80ul to start.  If after 5 minutes, the animal is still not completely under, then a small amount more can be injected.

 

  1. Immunization

Apply eye moisture salve to the eyes of anesthetized animals.

Shave the dorsal skin.  Use emery paper to lightly abrade it.  Hydrate with a wet cotton swab.

Apply a mixture of antigen plus adjuvant in cream, using a pipette and cotton swab.

Cover the area with dermal adhesive tape (DuoDerm extra thin).

 

A subcutaneous administration of antigen plus adjuvant is usually used as a control.  For this you do not need to anesthetize or shave.  Use a 26 g needle.