Materials

ISH-labeled sample with one biotinylated and one digoxigenin-labeled probe (unit 8.3) PBS (appendix 2a)/0.05% Tween 20/2% normal goat serum 1:2000 mouse anti-digoxin (Sigma) in PBS/0.05% Tween 20/2% normal goat

PBS (appendix2a)/0.05% Tween 20

1:25 alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (Dako) in PBS/0.05% Tween 20/2% normal goat serum

PBS (APPENDIX 2A)

New Fuchsin reagent (see recipe) 4x SSC (appendix 2a)/0.05% Tween 20

1:50 horseradish peroxidase-conjugated avidin (HRP-avidin; Dako) in 4x SSC

(appendix 2A)/5% nonfat dry milk 3,3'-diaminobenzidine (DAB) solution (see recipe) 20% hematoxylin (Sigma) in H2O 50%, 70%, 96%, and 100% ethanol 100% xylene

Entellan (organic-based mounting medium; EM Science)

24 x 50-mm coverslips Coplin jars

NOTE: All buffers containing nonfat dry milk, normal goat serum, or antibodies should be prepared fresh.

1. Block ISH-labeled sample with 100 |l PBS/0.05% Tween 20/2% normal goat serum under a 24 x 50-mm coverslip 10 min at 37°C.

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