Posthybridization for Peroxidase Based Color Development

1. Wash slides in IX PBS twice for 5 min each time.

2 Add 10 juL of streptavidin-peroxidase complex (100 pg/mL in PB S pH 7 2) Gently apply the coverslips

3 Incubate slides at 37°C for 1 h

4 Remove coverslip, wash slides with IX PBS twice for 5 min each time

5 Add to each well 100 pL of 3'-amino-9-ethylene carbazole in the presence of 0 03% hydrogen peroxide in 50 mM acetate buffer (pH 5 0)

6 Incubate slides at 37°C for 10 min to develop the color—this step should be carried out in the dark After this period, observe slides under a microscope If color is not strong, develop for another 10 min.

7 Rinse slides with tap water and allow to dry.

8 Add 1 drop of 50% glycerol in PBS and apply the coverslips

9 Analyze with optical microscope—positive cells will be stained a brownish red.

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