Perform sequential TSA detection

4. Incorporate the first HRP into the ISH signal (units 8.3 & 8.4).

Note that although two differentially labeled probes are simultaneously hybridized, one label is used to incorporate HRP followed by afluorochrome tyramide reaction, after which the second label is used to incorporate HRP followed by a different fluorochrome tyramide reaction. For example, using both biotin- and digoxigenin-labeled probes together, strep-tavidin-HRP is incorporated and a cyanine 3 tyramide reaction is performed first. This is followed by an anti-digoxigenin-HRP incorporation and a fluorescein tyramide reaction. An HRP-inactivation step (detailed in step 10) is performed between the HRP incorporations to prevent carryover tyramide binding.

5. Wash three times, 15 min each, in 4x SSC/0.1% Triton X-100 at room temperature.

6. Wash 5 min in 4x SSC at room temperature.

7. Prepare the first working tyramide solution (see Basic Protocol 3, step 2) and treat cells as described (see Basic Protocol 4, step 15a or 15b).

8. Wash three times, 15 min each, in 4x SSC/0.1% Triton X-100 at room temperature.

9. Wash 5 min in 2x SSC at room temperature.

10. Inactivate the first HRP by adding H2O2 to sodium acetate/sodium azide buffer at a final concentration of 0.3% (v/v), placing 100 to 300 Ml on the slide or coverslip, and incubating 15 min at room temperature. Wash three times, 5 min each, in either PBS or 2x SSC.

The H2O2 must be added to the sodium acetate/sodium azide buffer immediately before it is used.

11. Rinse twice, 5 min each, with 2x SSC at room temperature.

12. Repeat steps 4 through 9, using an HRP conjugate compatible with the second label (step 4) and a differently labeled tyramide (step 7).

13. Dehydrate, mount, and counterstain the slide (units 8.3 & 8.4).

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