Hi all,
I have no experience with CARD-FISH. But right now I suffered a lot from the autofluorescence of the sponges symbionts, which I tried to target. The autofluorescence was strong and universal. What's worse, the de-fluorescence methods didn't work well. So I am thinking about CARD-FISH.
My samples were collected in June 2013. Some of them were immediately fixed by 4% paraformaldehyde while some of them were fixed by RNA later. All samples were restored under -20/80 centigrade. From my own opinion, none of these samples should be used for CRAD-FISH now.
Any input is welcome.
Cheers! Fang
Hi Fang,
fixing samples in 4% FA is fine for downstream CARD-FISH analysis. The main question is if you stored your samples in any kind of solution (e.g PBS/EtOH) that prevents the sample from freezing. If yes, I don´t see any problem.
Can´t tell you anything on RNAlater & CARD-FISH though.
Cheers, Hannes
Hi Fang,
We sometimes freeze bacterial cells in 2% FA directly after fixation when we do not have the time for immediate downstream processing. We keep these samples then for several months at -80C and they work excellently for FISH/CARD-FISH analyses. So your samples may still work for CARD-FISH, although the ultrastructure may not be the best anymore. So it depends on what kind of question you want to answer and if you need perfect ultrastructure. Also did you try HCl treatment to reduce autofluorescence?
Cheers,
Manuel
Hi Manuel,
The HCl treatment didn't work well. I think my main problem is to get the perfect ultrastructure as I am not visualising a group of bacteria from a biofilm. The bacteria cells were embedded in sponge tissues and formed cell strings. If I want to display that the cell strings consist of different bacterial taxa, the resolution of CARD-FISH has to be perfect then.
Cheers！
Fang
Hi,
I am performing FISH for the first time. My purpose is to analyze the bacterial diversity of environmental biofilm but my samples were stored at 4°C in PBS (for about 3 months). In your opinion can I use it?
Thank you very much
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