實驗概要
The method provides a 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.
主要試劑
1. Hydrogen chloride (HCl), 1 M and 2 M
2. Borate buffer 0. 1 M
3. Borax (sodium tetraborate) 38.1 g / liter, pH to 9.0
4. Phosphate-buffered saline (PBS), pH 7.4, with 0.1% triton X-100
實驗步驟
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.