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8. Apply 100 |l of 1:2000 mouse anti-digoxin under a 24 x 50-mm coverslip and incubate 30 min at 37°C.

9. Wash twice for 1 min each with 100 ml PBS/0.05% Tween 20 at room temperature with gentle agitation.

10. Apply 100 |l of 1:100 TRITC-conjugated rabbit anti-mouse IgG and incubate 30 min at 37°C.

11. Wash twice for 5 min each with 100 ml PBS at room temperature with gentle agitation.

12. Dehydrate by washing 1 min each in 100 ml of 70%, 90%, and 100% ethanol at room temperature. Air dry.

13. Mount in antifading mounting medium. Store preparations up to several months at 4°C in the dark.

A universal protocol for a double-target bright-field ISH using biotin- and digoxigenin-labeled repeat-sequence probes is described. This protocol is applicable to all previously described procedures for sample preparation. The targets are detected using alkaline phosphatase (red) and horseradish peroxidase (brown). The DNA is counterstained with hematoxylin (blue). For a more detailed discussion of bright-field detection methods for ISH, see unit 8.4.

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