•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 4oC in cold 4% paraformaldehyde.
•After 2 hours, rinse the tissue with PBS and store in 70% ethanol at 4oC. The tissue could also be stained with Carmine Alum overnight at this point (see Carmine Whole mount protocol).
•ß-Estradiol 3-benzoate, Sigma E-8515
[1,3,5-Estratriene-3,17 ß-diol 3-benzoate]
•Progesterone, Sigma P-0130
•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 (≤ 60oC) 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.
Make up at [3 mg/ml] in PBS or saline. Store aliquots at -20oC.
Inject 10 μl per gram of bodyweight.
1. Working in a fume hood, heat 450 ml of ddH2O to 60oC.
2. With stirring, add 20 g of paraformaldehyde powder to the heated water. Cover and maintain ≤ 60oC 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.