scientificprotocols authored about 3 years ago

I. BrdU Treatment

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.)

II. BrdU Detection

A. Perfusion fixed, paraffin sections:

  1. Deparaffinize sections using standard xylene/alcohol procedures.
  2. Treat sections with 0.1% Trypsin in 0.1M TBS (pH7.5) + 0.1% CaCl2 for 20 minutes at 37oC in a humid box.
  3. Rinse 2 x 3 min in PBS.
  4. Treat sections with 0.3% hydrogen peroxide for 10 minutes at room temperature.
  5. Rinse 2 x 3 min in PBS.
  6. Treat with 2N HCl for 1 hour at 37o C.
  7. Rinse 2 x 5 min in 0.1M borate buffer pH 8.5.
  8. Rinse 2 x 3 min in PBS.
  9. Treat with 0.2% Triton X-100 for 30 minutes at room temperature.
  10. Rinse 3 x 5 min in PBS.
  11. Block sections 1-2 hours at room temperature with PGB Superblock (see below) in humid chamber.
  12. Remove Superblock and incubate sections overnight at 4oC with anti-BUdR-POD antibody (Boehringer Mannheim Cat.# 1 585 860) diluted 1:15 in PGB diluent.
  13. Rinse sections 3 x 5 min in PBS.
  14. Incubate with unactivated DAB for 5 minutes followed by activated DAB for 10 minutes at room temperature in the dark.
  15. Rinse sections with distilled water and coverslip with an aqueous mounting media.

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.

III. Solutions for BrdU Staining

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.


Average rating 0 ratings