Hi,

Unfortunately I am not an expert of these protocols, I am more a modelling person. But maybe Lucas Tanguy (https://www.linkedin.com/in/tanguy-lucas-2351675b/?originalSubdomain=fr) one of my coauthors could help you, he worked a lot on drosophila Fish and Zelda.

I hope this help.

Jeremy,

I work with Drosophila ovaries and embryos. And I also use Stellaris probes. I too have noticed that when performing smFISH-IF, the added steps required for IF leads to considerable loss of smFISH signal. The RNA I am detecting is extremely abundant, and so even with IF, I can still visualize it—however, exposure settings on my scope have to be increased to see it, compared to when doing smFISH alone. that being said, I do use the probes at 100 ng/µl (i.e. 1 µl of 10 µM probe into 100 µl of hybridization buffer), which seems to be higher than most peoples' protocols in the first place.

Some things I have tried, without much success, are:

1. Using DEPC-treated water (though the probes are DNA, RNases could still effect the RNA targets with the increased number of incubations when adding IF to the protocol)

2. Store-bought, nuclease-free PBS solutions

3. Post-fixing after permeabilization steps (though I think this is more to improve the subsequent IF than it is to improve smFISH)

The one thing I have yet to try—because I haven't done the experiment in awhile—is to post-fix after washing the hybridized tissue (i.e., hybridize, wash, post-fix, proceed with IF protocol). This may help stabilize the duplexes, and the nucleic acids themselves, and improve signal. In fact, I have seen some protocol that post-fix after every single step! That is probably overkill.

In summary, if I were you, I would proceed with smFISH as normal, and after tasing steps, post-fix for 20-30', wash several times with PBST, and then proceed with your IF protocol. Also, if your probe concentration in your hybridization buffer is

Dear Ryan,

Thanks a lot for your advices, i recently tryied To put the Ab in the hyb buffer and this work pretty Well.

When you post fix after smfish you dont see decrease in fish signal? I was using a probe with Q670, i Will tryied with Q570 which is much more brighter.

I saw you are in the P Rangan so we could keep in touch to shared our expérience with immuno smfish in drosophila.

Thanks a lot

Hi Jeremy,

Did you had a look at the background intensity in the smFISH channel with and without the immuno stain? The reason Im asking is that the smFISH signal is tipically very low compared to an antibody stain, so even a little bit of bleed-through from the AB fluorescent channel could "hide" your smFISH signal.

We usually run the immuno after imaging the smFISH results. If you want you can remove the smFISH probes by washing 30 minutes with 65% formamide in 2X SSC.

Lars