Project protocol - Contents
- Workflow and sampling
- Equipment and supplies
- Reagents and solutions
- Procedure: BrdU preparation, administration, and detection
- Definitions and calculations
Workflow and sampling
Step Procedure Equipment Data collected 1 Mice injected with BrdU - - 2 Mice perfused and brains harvested - - 3 Brain immunohistochemistry Inverted microscope BrdU-labeled cells in the rostral migratory stream (RMS)
Equipment and supplies
- Superfrost/Plus microscope slides
- Paraffin (Paraplast Plus)
- Zeiss 200M Axiovert inverted microscope
- Axiovision software (v 4.6)
- NIH ImageJ (v 1.42)
Reagents and solutions
- BrdU (bromodeoxyuridine; Sigma Aldrich, St. Louis MO)
- Mouse anti-BrdU monoclonal antibody (BD Biosciences, Mississauga ON Canada)
- Biotinylated horse anti-mouse immunoglobulin (Vector Laboratories, Burlingame CA)
- VECTASTAIN Elite ABC kit (Vector Laboratories, Burlingame CA)
- Diaminobenzidine (Sigma Aldrich, St. Louis MO)
- Avertin (Sigma-Aldrich, St. Louis MO)
- Acetic acid
Procedure: BrdU preparation, administration, and detection
- Mice are injected with a single dose of BrdU (50 gm/kg body weight).
- One hour later, mice are anesthesized with Avertin (0.2 mL/10 g body weight) and perfused with acetic acid : 95% ethanol (1:3).
- Brains are removed from the skull and processed for paraffin embedding as previously described (Poon et al 2010).
- Each hemisphere is is serially sectioned in the sagittal plane (see Figure below) at 8 µm and then mounted on Superfrost/Plus slides.
- Sections are deparaffinized, rehydrated, and treated with 1M HCl for 30 min at 37°C.
- Slides are incubated with mouse anti-BrdU monoclonal antibody (1:200 dilution) overnight and then with biotinylated horse anti-mouse immunoglobulin (1:200dilution) for 1 h on the next day.
- BrdU immunoreactivity is revealed using the VECTASTAIN Elite ABC kit and 3, 3'-diaminobenzidine according to manufacturer's instructions.
- BrdU-labeled cells are counted under bright-field illumination and with the aid of a 20x objective inverted microscope (see Figure below).
- RMS length is measured using NIH ImageJ software.
- Linear density (number of BrdU+ cells per mm of RMS length) is calculated for each animal.
Modified from http://bioteaching.wordpress.com/2010/04/16/stem-cell-research/
- BrdU+ cells per mm RMS
Poon A, Li Z, Wolfe GW, Lu L, Williams RW, Hayes NL, Nowakowski RS, Goldowitz D. Identification of a Chr 11 quantitative trait locus that modulates proliferation in the rostral migratory stream of the adult mouse brain. Eur J Neurosci. 2010 Aug;32(4):523-37. doi: 10.1111/j.1460-9568.2010.07316.x.