Table 1.

Troubleshooting

Problem	Potential Solution(s)
High background signal	Use smaller volumes of conjugated antibodies or substrate. Try running multiple in situs with various amounts of antibody or substrate in parallel to see which volumes produce the best results.
Embryos stay suspended in buffer	This sometimes occurs in the first wash after posthybridization, especially in P. miniata. Try to remove as much hybridization buffer as possible before adding MOPS or maleic acid buffer (depending on species). Alternatively, pipet the suspended embryos into a larger container such as a 15mL Falcon tube and add additional MOPS or maleic acid buffer to dilute the hybridization buffer even further.
Embryos are sticky or lost	Double check your MOPS and/or maleic acid buffer to ensure enough Tween-20 was added. Not enough detergent may cause embryos to stick to pipet tips and thus be lost during washes.
No or low signal	In the case of low signal, try using more substrate or antibody. If there is no signal, see the previous section on using a positive control to determine whether the issue is with the probe or some other factor.
Damaged embryos or embryos with poor morphology	Damaged embryos may result from being too aggressive when pipetting or handling them. Ensure you are always gentle as it is very easy to damage fixed embryos and larvae. Embryos should never be centrifuged. If embryos still have poor morphologies, try leaving them an additional day or two in fixative solution before dehydrating to 70% EtOH.