Immunofluorescence staining – quantification of activated-caspase 3-positive cells in brain slices:

Immunofluorescence staining – quantification of activated-caspase 3-positive cells in brain slices:

1.                  Dissect out brains and post-fix in 4% PFA overnight at 4⁰C

2.                  Wash brains in PBS 0.01M 3x10 minutes

3.                  Cryoprotect brains in 30% sucrose

4.                  Using a cryostat (Leica, Wetzlar, Germany), perform 50 µm coronal sections

5.                  Remove PBS and add blocking solution for 2 hours at room temperature (RT)

6.                  Remove blocking solution and add primary antibody diluted in antibody dilution buffer overnight at 4°C

7.                  Wash slices in PBS 0.01M 3x5 minutes

8.                  Incubate slices with DAPI and DyLight488-coupled secondary antibody diluted in secondary antibody dilution buffer at RT for 2 hours

9.                  Wash slices in PBS 3x5 minutes

10.              Mount sections on to slides using fluoromount and secure with cover glass

Solutions:

30% sucrose solution: 30 g sucrose in 100 ml distilled water

Blocking solution: 7% normal donkey serum/0.3% triton diluted in PBS 0.01M

Primary antibody dilution buffer: 2% bovine serum albumin/0.05% azide/0.1% triton diluted in PBS 0.01M

Secondary antibody dilution buffer: 2% bovine serum albumin/0.05% azide diluted in PBS 0.01M

List of Reagents:

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