I am currently developing a rapid method which involves dual FISH staining of water microbes, the FISH method I am using is a well established method in our lab and we have developed a couple of protocols for the detection of E. coli and Salmonella spp. With these developed methods a fixation step wasn't required, however with this new method for water microbes I find that a fixation step is necessary to achieve sufficient hybridization. The issue I now run into is that in one spectrum (red imaging field) with an Alexa probe, I see sufficient hybridization. But in the other spectrum (green/blue imaging field) with a FAM probe, the signal detected is very weak. What is interesting is when I include a paraformaldehyde step in the E.coli & Salmonella protocols, a similar observation is observed (also with a green Alexa probe). Does anyone have any advice to offer of how to improve this or can detail why such an effect is happening?

I currently fixate with 3% paraformaldehyde in PBS for 1H at 4C, and wash with PBS.

Many thanks!