1. Block for 30 min in 20% sheep serum in PBS at room temperature.

2. Incubate with primary antibody/antiserum at the desired dilution in PBS plus 5% sheep serum for 1-2 h at room temperature.

3. Wash several times with PBS.

4. Incubate in biotinylated secondary antibody at appropriate dilution in PBS.

6. Incubate in alkaline phosphatase streptavidin (1/100) in PBS for 30 min at room temperature.

8. Incubate in Fast Red in the dark until desired reaction product intensity is achieved (15 min-2 h). Stop the reaction by washing in water.

9. Mount in Mowiol with a coverslip.

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