Has anyone done FISH on cre-recombinased mice? I want to know if anyone has successfully been able to quench the autofluorescence from the those mice to successfully visualize the targeted genes? I have cre-mice crossed with ROSA26 tomato mice that I would like to do FISH on.
Any help or guidance would be greatly appreciated. Thank you!!!!
Hi Sana,
I do a lot of FISH on free-floating brain slices from mice that express a fluorescent molecule after cre-mediated recombination. Since my goal is to detect mRNA and I'm using long RNA probes, the hybridization temperature is always at around 70°C, overnight in hybmix. Under these conditions, the endogenous fluorescence of fluorescent molecules is always lost (with either GFP, Zsgreen1 or mCherry).
In addition, I have also noticed that, when using viruses expressing mCherry, the fluorescence was lost after a 30min-incubation in 3% H2O2 diluted in PBS. I don't know whether it would work for tdtomato as well but you may want to try (if your protocol doesn't already have a step like that).
If increasing the hybridization temperature is not possible in your protocol, maybe you could try including a seperate step of high temperature incubation before doing the actual hybridization, and see if you can get rid of the endogenous fluorescence.
Good luck!
I have down ISH on foxg1 cre mice. In the future, i am interested performing Fish. With fish I found it difficult to get two probes to bind at higher temperature. Good luck getting it to work!
Hi Chrisopher,
I had the same problem with a couple of probes that worked separately but not together. I solved the problem by increasing the dithiothreitol (DTT) concentration in the hybridization mix up to 1M. Maybe this can help...