In order to determine whether the brain is undergoing neurogenesis, scientists needed to devise a way to mark proliferating cells. One way to do this is to identify cells that are in the S phase – also known as the “synthesis” phase – of replication. Here, new copies of DNA are synthesized so that the cell may eventually split into two daughter cells, each with a copy of DNA. Originally, scientists used Tritiated Thymidine, which is a radioactive form of the nucleotide Thymidine, to measure cells in the S phase. However, technical difficulties (see BrdU vs. Tritiated Thymidine) spurred scientists to explore other options, leading us to the more modern technique of BrdU staining. BrdU is a synthetic analogue of Thymidine that is also incorportated into cells during DNA replication, which allows us to successfully identify dividing cells. Essentially, BrdU provides scientists with a way of staining cells that are proliferating in the human brain.
*Image from: http://en.wikipedia.org/wiki/BrdU
How does it work?
When cells are synthesizing new copies of genetic material during S phase, they pull nucleotides from the intracellular environment to create a strand of DNA. When a scientist injects BrdU into the bloodstream of a test animal, the chemical becomes available to all cells – most importantly, those which are proliferating. As the cells build their new genetic strand, they pull either Thymidine nucleotides or BrdU analogues from their environment. Since the two chemicals are analogous, some spots of the genetic code that call for Thymidine will instead receive BrdU. The special thing about BrdU is that it can be stained with an antibody using immunohistochemical methods, whereas Tritiated Thymidine is only detected by radiographic techniques. The antibody used in this case will recognize and stain for cells that have incorporated BrdU into their genetic code. Any cell that stains positive for BrdU can be said to have undergone proliferation after the time of injection.
BrdU vs. Tritiated Thymidine
The most important reason BrdU has become the predominant method to identify proliferating cells is its efficiency. Whereas the Tritiated Thymidine method may take 3-12 weeks to develop a picture, BrdU only takes 1-3 days. Furthermore, BrdU methods only require tissue section of approximately 30 micrometers, while Tritiated Thymidine requires sections of 2-3 micrometers; this allows scientists to reduce the number of slides to be analyzed. Overall, BrdU allows for a significantly more efficient and cost effective method of identifying cells undergoing proliferation.
Use of Bromodeoxyuridine-immunohistochemistry to Examine the Proliferation, Migration, and Time of Origin of Cells in the Central Nervous System. Michael W. Miller et. al.
BrdU Immunohistochemistry for Studying Adult Neurogenesis: Paradigms, Pitfalls, Limitations, and Validation. Philippe Taupin.
What does it look like?
Here are some images of BrdU staining. The first image shows a BrdU staining of nuclei, while the second image shows a BrdU staining co-labeled with a Lamin stain.
Page by: Alex Crespo