Preparing Steroids (Testosterone/Estrogen) for injecting Mice

Advance Animal Physiology

 

 

Materials Needed

250 mL Pure Corn Oil

8 mg β-estradiol 3-benzoate

8 mg Testosterone

2 x 100 mL sterile glass bottles with lids w/ septum

3 x 1 L bottles with lid

Aluminum Foil

Autoclave tape

2 spatulas, used for steroids only

Thick latex gloves

Mask with filter

2 cases of BD microfine Syringes 0.5 cc

Sharps container

3 packages of Q-tips

1 L of isopropyl alcohol

Labeling tape (pink, blue, white and yellow)

Marker

Transporting bin

Lab coat

 

 

 

Warnings!!

These steroids are highly carcinogenic and will easily absorb into the skin. Do not handle without proper protection (lab coat, mask and gloves). Women who are pregnant or breastfeeding should not handle the steroids at all.

 

Prepare the steroids under a fume hood where NO OTHER steroid assays (RIA, EIA) are likely to be performed. Any contamination can alter assays that measure steroids.

 

Step 1: Autoclaving materials and sterilizing corn oil

 

  1. Place ~300 mL of pure corn oil into the two 1L bottles. Place lids on top but DO NOT screw the cap on tightly. Place the aluminum foil over the lids then use the autoclave tape to attach each side of the tape to the bottle.
  2. Properly autoclave the two 100 mL bottles, with there lids loosely screwed on, tin foil and autoclave.
  3. Use a steri-pack to wrap the 2 spatulas.
  4. Autoclave all the materials. Place a pan underneath the corn oil bottles in case of spilling
  5. After autoclaving, the oil will be extremely HOT, so open the autoclave door and allow a few hours to cool

 

Step 2: Preparing the steroids in the vehicle solution

  1. Place 80 mL each of the sterile, cooled corn oil into 3 100 mL sterile bottle, close lid.
  2. Label one bottle: Date, “Sterile Pure Corn oil, 100 µL per adult mouse” and your initials.
  3. Using the designated spatula, measure (using the microscale) 8 mg (0.0080 g) of Estradial and carefully place into the bottle. Screw cap tightly. Vortex, then label using the tape and marker. Put the date, “β-estradiol 3-benzoate, 8 mg/80 mL (50µg/mL), 100 µL per adult mouse (5µg)” and your initials.

 

Store indefinitely at room temperature.

 

  1. Using the designated spatula, measure (using the microscale) 8 mg (0.0080 g) of Testosterone and carefully place into the bottle. Screw cap tightly. Vortex, then label using the tape and marker. Put the date, “Testosterone CIII, 8 mg/80 mL (50µg/mL), 100 µL per adult mouse (5µg)” and your initials.”

 

The testosterone is a CIII, so it is a controlled substance. It needs to be stored at room temperature in a locked safe.

 

Step 3: Preparing the steroids for injections

  1. Prepare the syringes weekly. Unwrap 168 BD syringes.
  2. After gently vortexing the estrogen in solution, draw 100 µL each in 56 syringes. CAREFULLY put the orange caps back on. Label the syringes with pink labeling tape. Store the syringes (room temperature) in a plastic, disposable cup and lid that is also labeled.
  3.  After gently vortexing the testosterone in solution, draw 100 µL each in 56 syringes. Label the syringes with blue labeling tape. Store the syringes in a plastic, disposable cup and lid that is also labeled. These will also need to be stored in the safe.
  4. Draw 100 µL  of the sterile pure corn oil each in 56 syringes. Label the syringes with yellow or white labeling tape. Store the syringes in a plastic, disposable cup and lid that is also labeled.

 

Step 4: Administering the sub Q injections

 

  1. Place the prepared syringes in the small test tube order by  cage treatments. So if cage one is to receive testosterone, place two syringes of testosterone (blue). If cage two is to receive estrogen, then 2 pink syringes, and etc…)
  2. Between 8-10 am each morning, the injections need to take place.

 

This will take place in the mouse room. PLEASE be as quiet as possible. Do not shout or over handle the mice. Each injection should take no longer than 45-60 seconds to perform. A partner can aid in preparing the mice and cages, and placing the cages back. Please do not wear strong perfumes during this procedure.

  1. Properly restrain each mouse.
  2. Rub the injection site using a Q-tip with rubbing alcohol, then give the subcutaneous injection. Put mouse immediately back in the cage, and repeat for its cage mate. If  mouse B’s marking has faded, use the sharpie and remark the mouse.
  3. Throw away the syringes in the sharps container immediately and DO NOT recap the syringes.
  4. Place the cage back in its place.
  5. On Weds, the weights will be recorded.