In situ hybridization and antibody labeling

Immunolabeling protocol

1:         Fix fish in 4% PFA (with 0.25% triton) at room temp for 2 hr OR 4 degrees overnight; rocking

2:         Wash 2X 5 minutes in 1X PBS, 0.1% triton

3:         If over 3dpf, wash overnight in hyclone water at room temp rocking

4:         Block in AB block (recipe in protocol book; stock at 4 degrees), with 5% goat serum at room temp for 1-4hrs

5:         Incubate in primary antibody overnight in AB block+5% goat serum, 4 degrees, rocking

                           -Antibody concentration depends on the antibody

6:         Wash 3X 15 minutes (at least) in 1X PBS, 0.1% triton at room temp rocking

7:         Incubate in AlexaFluor secondary antibody 1:1000 in 1X PBS, 0.1% triton overnight at 4 degrees rocking - --  (wrap in foil as the secondaries are fluorescently labeled)

8:         Wash 3X15 minutes in 1X PBS, 0.1% triton at room temp rocking

9:         Sink in 50-60% glycerol in 1X PBS, 0.1% triton and store at 4 degrees no more than a week prior to imaging.

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