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BrdU staining protocol

Kindly submitted by Daniel Webber, King's College London.

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Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) is a synthetic analog 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 culture 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 3 times, 5 min per wash.
  6. Continue with standard staining procedure as described in our other IHC 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 60°C 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 37°C for 10 min, and washed with cold PBS 3 times for 5 min.

Examples


Fig 1: BrdU protocol in vitro
In vitro dividing cells can be labeled with BrdU and following the protocol were visualized with HRP conjugated secondary antibodies and DAB to produce a nuclear staining pattern.  Scale bar = 100 µm.



Fig 2: BrdU protocol in vivo
In vivo cells can be labeled either by injecting BrdU or alternatively label the cells prior to transplantation and then identify them using the protocol.  Neural stem cells were labeled 48 hr prior to transplantation into a spinal cord injury at cervical level 4. The cells were identified throughout the dorso-ventral axis of the cord and had also begun to migrate rostro-caudally from the injection site. They were visualized using the described protocol, followed by a TRITC conjugated secondary antibody. Scale bar = 500 µm.

Fig 3: BrdU protocol in vivo
In vivo cells were labeled prior to transplantation and then identified using Abcam Polyclonal anti-BrdU with a TRITC conjugated secondary antibody. Scale = 100 µm.

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. J Immunol, 107(1):79-88.
  • Chu K, Kim M, Chae SH, Jeong SW, Kang KS, Jung KH, Kim J, Kim YJ, Kang L, Kim SU, Yoon BW (2004) Distribution and in situ proliferation patterns of intravenously injected immortalized human neural stem-like cells in rats with focal cerebral ischemia. Neurosci Res, 50:459-465.
  • Gage FH (2000) Mammalian neural stem cells. Science 287:1433-1438.
  • Hahn CG, Han LY, Rawson NE, Mirza N, Borgmann-Winter K, Lenox RH, Arnold SE (2005) In vivo and in vitro neurogenesis in human olfactory epithelium. J Comp Neurol, 483:154-163.
  • Matsuura, Sachiko.  Suzuki, Kazuo (1997); Immunohistochemical Analysis of DNA Synthesis during Chronic Stimulation with Isoproterenol in Mouse Submandibular Gland. J Histochem Cytochem, 45(8):1137-1145.
  • Nieoullon V, Belvindrah R, Rougon G, Chazal G (2005) mCD24 regulates proliferation of neuronal committed precursors in the subventricular zone. Mol Cell Neurosci, 28:462-474.
  • Tischler, AS (1995); Triple immunohistochemical staining for bromodeoxyuridine and catecholamine biosynthetic enzymes using microwave antigen retrieval. J Histochem Cytochem, 43(1):1-4.

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