I was wondering if anyone has been able to successfully perform phallodin staining with hcr in situ hybridization in whole-mount tissue. For my samples, which are 5 day old zebrafish embryos, I have been trying to get phalloidin staining to work with my hcr probe and a nuclear counter stain. I have optimized the protocol to successfully get the nuclear counter stain and probe to work, but I can never get the phalloidin staining to work. I am using methanol free 4% PFA and dehydrating my embryos in 70% ethanol rather than methanol. Additionally, I am not performing a bleaching step or using acetone so that the F-actin is not degraded. I perform the phalloidin counterstain after the last step of the standard hcr protocol and use 1% BSA as block prior to using the phalloidin stain which is also in block.

I was wondering if anyone has successfully performed hcr with a phalloidin counterstain. I have seen some papers that have mentioned using the two together but the staining never looks overly clear. I am unsure if the probe, wash, or amplification buffers used in hcr contain methanol in them as the product ingredients are not listed.

Thanks for help.