Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: BrdU preparation, administration, and detection
• Definitions and calculations
• Data

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
• Ethanol

Procedure: BrdU preparation, administration, and detection

1. Mice are injected with a single dose of BrdU (50 gm/kg body weight).
2. One hour later, mice are anesthesized with Avertin (0.2 mL/10 g body weight) and perfused with acetic acid : 95% ethanol (1:3).
3. Brains are removed from the skull and processed for paraffin embedding as previously described (Poon et al 2010).
4. Each hemisphere is is serially sectioned in the sagittal plane (see Figure below) at 8 µm and then mounted on Superfrost/Plus slides.
5. Sections are deparaffinized, rehydrated, and treated with 1M HCl for 30 min at 37°C.
6. 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.
7. BrdU immunoreactivity is revealed using the VECTASTAIN Elite ABC kit and 3, 3'-diaminobenzidine according to manufacturer's instructions.
8. BrdU-labeled cells are counted under bright-field illumination and with the aid of a 20x objective inverted microscope (see Figure below).
9. RMS length is measured using NIH ImageJ software.
10. 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/

Definitions and calculations

• RMS: rostral migratory stream
• BrdU: bromodeoxyuridine

Data collected by investigator

• BrdU+ cells per mm RMS