Mice were injected i.p. with 0.01 mL/g bromodeoxyuridine (Amersham Cat.# RPN 201). Animals were sacrificed two hours later and perfusion fixed with 4% paraformaldehyde/PBS. Mammary glands were removed, post fixed in 4% paraformaldehyde overnight then paraffin embedded and sectioned and mounted onto slides. Alternatively, mice were sacrificed and the glands removed immediately without fixation and frozen for cryostat sectioning. Not surprisingly, the morphology is best on the paraffin sections. (Attached photomicrograph shows a labeled duct in the abdominal gland from a post-pubertal mouse.)
A. Perfusion fixed, paraffin sections:
For fresh frozen, cryostat sections:
Take slides from -70 and immediately postfix for 10 minutes in 4% paraformaldehyde. Rinse in several changes of PBS and procede with the above procedure beginning with step 4. Note: the trypsin treatment will tear up your tissue and is not necessary. Also, reduction of the HCl treatement to 30 min is sufficient for good staining and will leave the sections in better shape.
PGB Superblock for 100 mL Final Conc. BSA 10g 10% NaN3 0.5 mL of 10% 0.05% Normal Goat Serum 10 mL 10% 0.5M PO4 (pH 7.6) 79.5 mL PGB Diluent for 50 mL PGB Superblock 10 mL Triton X-100 100mL PBS 40 mL DAB Solution DAB Stock - 3'-3' diaminobenzadine, 2.5 mg/mL in 0.1M PO4, pH7.6. Unactivated DAB - 1 mL DAB + 19 mL 0.1M PO4 +450 mL 1% aq. NiCl2, pH7.6. Activated DAB - 10 mL unactivated DAB + 23 mL 30% H2O2.