BrdU In-Situ Detection Kit From BD Biosciences

BrdU, or 5-bromo-2-deoxyuridine, is a synthetic analog of the nucleotide thymidine that is incorporated into a cell’s DNA when it is going through cell division, particularly the S-phase of the cell cycle. Because of its similarity to thymidine, BrdU is used in many animal research studies that focus on cell division, proliferation and viability. In our studies, BrdU (5 mg/kg body weight) is injected intravenously into an animal 4 hours before necropsy to allow time for its incorporation into DNA so that it can be subsequently detected in tissue through immunohistochemical methods.

The BrdU In-Situ Detection Kit from BD Biosciences detects BrdU that has become incorporated into DNA during the cell cycle; the kit can be bought with enough reagents to assay either 50 or 200 slides. The kit is based on the premise that an antigen retrieval system labels cells that have incorporated BrdU. In our studies, we removed intestinal tissue from an animal and immediately placed it in formalin for preservation. Subsequently, the tissue underwent standard tissue processing such as dehydration, clearing and perfusion with paraffin. Embedded tissue was sliced using a microtome and sections were placed on microscope slides. It is important to note however, that this kit can also be used on frozen tissue sections, or cultured cells on slides. The technical information included with the kit states that it is specific against mouse, human and rat species; however, we have used this kit with porcine tissues and it has been very effective.

Because the BrdU staining procedure is more rigorous than some other methods, such as H&E staining, it is relatively easy for tissue sections to come detached from the slide during processing, especially during the antigen retrieval step. Therefore, it is necessary to buy pre-coated slides or sub slides in your own laboratory. A simple chrome-potassium-gelatin subbing solution works fine. The brown stain obtained after using the BrdU kit with a DAB buffer and chromagen indicates the cells in which BrdU incorporation has occurred. However, if using a gelatin-based subbing substrate, it is important not to incubate your slides with the DAB buffer and chromogen too long as this can increase background staining.

I recommend this kit because it was competitively priced and relatively easy to use. In addition, the kit contains mouse intestinal control slides that have been paraffin-embedded, which you can process alongside your samples of interest. If you do not obtain ideal staining in your tissue sections, you can compare your test samples with the controls included in the kit and determine whether there was an issue with your sample preparation or a mistake in carrying out the procedure. The kit is straightforward, minimal additional materials are needed, and a contrasting counter stain, such as hemotoxylin can be used very effectively. We plan to continue using this kit in all of our future studies that require measures of cell proliferation in our porcine model of intestinal injury and adaptation.