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BrdU Immunohistochemical Staining Protocol Description To measure

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					BrdU Immunohistochemical Staining Protocol

Description

To measure DNA synthesis or cell proliferation, 5-bromo2’-deoxy-uridine (BrdU) can be
incorporated into DNA in place of thymindine. Cells, which have incorporated BrdU into
DNA, can be quickly detected using a monoclonal antibody against BrdU. The binding of
the antibody is achieved by denaturation of the DNA. This is usually obtained by
exposing the cells to acid, or heat. This protocol described a method to assess neuronal
migration in mice.

Reagents

Primary Antibody: Rat Anti-Human BrdU (Clone BU1/75, ICR1) (Accurate Chemical &
Scientific, Cat# OBT0030). Optimal dilution 1:300. Species Reactivity: Human, mouse,
rat (refer to antibody datasheet for more information).

Secondary Antibody: Rabbit Anti-Rat IgG (H+L), biotinylated (Vector Laboratories,
Cat# BA-4001). Optimal dilution 1:500.

Detection Reagent: HRP-Streptavidin (Vector Laboratories, Cat# SA-5004). Optimal
dilution 1:500

Preliminary

   1. Set up timed matings. Day of plug is deemed to be 0.5 days.
   2. Inject BrDU 50ug/ml at specific embryonic day.
   3. Kill pups at P0, and emersion fix in 4% PFA, or allow to develop to 8 weeks, and
      perfuse.

Slicing

   1. Mount brains in OCT and cut 14 micron sections on a cryostat.
   2. Dry sections overnight to adhere to slides.

Staining

   1. Place sections in Citrate Buffer (0.01M) Ph = 6.0. To make citrate buffer use
      2.94g of trisodium citrate in 1L. Adjust Ph with citric acid. These should be
      placed in the glass slide chambers.
   2. Heat up sections to about 90 degrees. When temperature reaches 90 degrees,
      remove slides, and cool for 10-15 mins.
   3. Wash twice for 5 mins in PBS.
   4. Once cool, block endogenous peroxidase with 2% H202, 5% methanol in water for
      5 mins. Make this up immediately beforeahand. To make up 300ml use 10ml
      H202 (30%), 15ml methanol 255 water). Prepare water bath at 37 degree for
      trypsin digest.
   5. Wash twice for 5 mins in PBS.
6. Then digest in trypsin (0.0125%) in water (36mg in 300ml), plus 0.05% CaCl2
    (150mg in 300ml) at room temp for 7 mins at room temperature, while gently
    shaking. Solution should be prewarmed at 37 degree before adding slides. (** NB
    tissue degraded – used 27mg of trypsin in 300ml 7/11/06 and worked well).
7. Wash three times for 5 mins in PBS. IMPORTANT WASH.
8. Then denature with 2N HCL for 30 mins at 37 degrees. This step is necessary to
    denature the DNA.
9. Wash three times for 5 mins in PBS.
10. Immediately apply primary antibody (1:300, 0.3% triton, 2% normal rabbit
    serum). The secondary antibody was raised in rabbit. Incubate overnight.
11. Wash 3x in PBS.
12. Apply secondary antibody (1:200) in 0.2% triton/PBS with 2% normal rabbit
    serum. Secondary antibody is anti-rat from elite kit. Incubate for 1 hours for 14
    um sections.
13. Wash 3x PBS
14. Make up ABC in 0.2% triton/PBS. To make up 10ml add three drops of reagent
    A, and three drops of reagent B. Incubate for 1 hour.
15. Wash 3x PBS.

				
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