hormone_BrDU ( Click to Download )

•Bed the animals on SaniChips, rather than corn pellets, to prevent any interference from phytoestrogens for 5-7 days before the hormone treatment.

•Two days before sacrifice, inject mice with 100 μl of E+P under the skin between the shoulder blades. [The final dose given is 1 μg E2 and 1 mg P]

•Two hours before sacrifice, inject the mice with BrdU intraperitoneally (10 μl per gram of body weight….ie: 20 gram mouse would get 200 μl).

•Remove the mammary glands, spread on parafilm paper squares, and place in tissue cassettes. Fix for 2 hours at 4^oC in cold 4% paraformaldehyde.

•After 2 hours, rinse the tissue with PBS and store in 70% ethanol at 4^oC. The tissue could also be stained with Carmine Alum overnight at this point (see Carmine Whole mount protocol).

Reagents:

Estrogen+Progesterone:

•ß-Estradiol 3-benzoate, Sigma E-8515

[1,3,5[10]-Estratriene-3,17 ß-diol 3-benzoate]

•Progesterone, Sigma P-0130

[4-pregnone-3,20-dione]

•Sesame oil, Sigma S-3547

E2 stock: [10 mg/ml] stock in sesame oil

10 mg E2 powder in 1 ml sesame oil.

Stir with mild heat (≤ 60^oC) until dissolved.

E+P for injections: [10 μg/ml] E2 Benzoate + [10 mg/ml] Progesterone in sesame oil

100 mg progesterone powder

10 μl of [10 mg/ml] E2 stock

10 ml sesame oil

Stir until dissolved.

BrdU: BromodeoxyUridine: Amersham RPN201 or Sigma B-5002

Protocol:

Make up at [3 mg/ml] in PBS or saline. Store aliquots at -20^oC.

Inject 10 μl per gram of bodyweight.

4% PFA:

1. Working in a fume hood, heat 450 ml of ddH₂O to 60^oC.

2. With stirring, add 20 g of paraformaldehyde powder to the heated water. Cover and maintain ≤ 60^oC on a hot plate.

3. Add 5 drops of 2N NaOH (1 drop per 100 ml). The solution should clear within a couple of minutes (There will be some fine particles that will not go away). Do not heat solution above 70°C.

4. Remove from heat and add 50 ml of 10X PBS. Adjust pH to 7.2; you may have to add some HCl. Filter and place on ice.