I am trying to visualize bacteria transfected with GFP tagged proteins growing in bone tissue. The bone has a high level of auto-fluorescence so I was thinking of demineralizing it, but I am not sure of the effect it would have on the bacteria and GFP. Has anyone had experience with fixing GFP bacteria in tissue?
Hi Vahid,
Maybe you could extend a bit your question:
What do you mean by fixing?
What do you want to measure?only bacterial fluorescence?
Hi Jacopo, thank you for your reply.
My goal is to visualise the bacteria, their pattern of distribution and numbers of the bacteria. That is why I used the fluorescent bacteria. But I'm not concerned with the bone tissue loss (if it is lost during demineralization and fixing stage).
I was thinking of fixing the tissue and demineralizing it to eliminate the auto-fluorescence and try to detect the bacteria again with some stain or marker, although I'm not sure if this will work or it is the best way to achieve the primary goal.
Unfortunately I have no clue whether demineralizing the bone tissue will help but maybe you could try the following things to enhance the signal from the bacteria:
- get an emission filter for GFP if you don't have one. If you take a bandpass centered at the emission of GFP you should cut off part of the autofluorescence spectrum (hoping it is not overlapping too much)
- illuminating with a 488nm laser (if you are not already doing it) will sharpen the emission. If you are using a lamp, then its spectrum will be broader and you will get undesired signal from everywhere in the sample.
about fixation, I usually use 30 minutes 4% paraformaldehyde at room temperature which has been proven to be one of the best fixation protocols.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3181414/
Also 2.5% glutaraldehyde or 10% formalin preserve well the cell integrity.
Hope this can help!
Best,
Jacopo
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