BrdU immunostaining procedure for cell cultures and tissue sections.

Protocol and tips for BrdU staining in tissue sections.

Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) is a synthetic analogue of the nucleoside thymidine. It is incorporated into replicating DNA in dividing cells and can be subsequently detected by anti-BrdU antibodies.

Immunostaining of the labeled cells is a standard IHC/ICC procedure but requires a step to denature DNA, allowing the antibody access to the BrdU. The procedure can be applied to cell cultures as well as tissue sections. Hydrogen chloride is a popular choice for the denaturing agent. The following procedure is an abbreviated version of the protocol linked on the datasheet for BrdU antibody - Proliferation Marker (ab1893), provided courtesy of D. Webber.

Materials and reagents

Hydrogen chloride (HCl), 1 M and 2 M

Borate buffer 0. 1 M   
Borax (sodium tetraborate) 38.1 g / liter, pH to 9.0

Phosphate-buffered saline (PBS), pH 7.4, with 0.1% triton X-100

Method

  1. Sections of paraffin-embedded tissues should be de-waxed before proceeding. An antigen retrieval step should be applied if recommended on the datasheet for the antibody, and may remove the need for an additional step to denature DNA using acid or other reagent, as discussed in the Tischler (1995) reference listed below. Frozen tissue sections and cell cultures or cytospins should be fixed in either paraformaldehyde or cold acetone or methanol.
  2. Incubate in HCl (1 M) for 10 min on ice to break open the DNA structure of the labeled cells.
  3. This is followed by HCl (2 M) for 10 min at room temperature, then 20 min at 37°C.
  4. Immediately after the acid incubations, neutralize by incubating the samples in borate buffer (0.1 M) for 10 min at room temperature.
  5. Wash in PBS (pH 7.4), 0.1% triton X-100 three times, 5 min per wash.
  6. Continue with standard staining procedure as described in the immunohistochemistry and cytochemistry protocols.

The acid incubation may affect the labeling of other antigens in multiple labeling; this will depend on the antigen and the antibody used to label it. Campana et al. (1988), discusses an alternative to the above procedure for double and triple staining of BrdU, Ki67, and TdT using NaOH instead of HCl to denature DNA.

Another modification, used in Matsuura et al. (1997), includes digestion of nuclear protein in resin-embedded sections. Sections were treated with 2 M HCl at 60oC for 15 min and washed in water for 15 min. After dehydration in an ethanol series, the sections were etched with xylene (15 min, twice), digested with 0.025% protease in PBS at 37oC for 10 min, and washed with cold PBS three times for 5 min.

References

Campana, D., Coustan-Smith, E., Janossy G. (1988); Double and triple staining methods for studying the proliferative activity of human B and T lymphoid cells. Journal Immunol Methods, Volume 107(1):79-88.

Matsuura, Sachiko.  Suzuki, Kazuo (1997); Immunohistochemical Analysis of DNA Synthesis during Chronic Stimulation with Isoproterenol in Mouse Submandibular Gland. Journal of Histochemistry & Cytochemistry, Volume 45(8):1137-1145.

Tischler, AS (1995); Triple immunohistochemical staining for bromodeoxyuridine and catecholamine biosynthetic enzymes using microwave antigen retrieval. J Histochem Cytochem. 43(1):1-4.


D. Webber
Professor McMahon's Laboratory
CARD Institute
King's College London
London, UK