Hi all,
Last month I have performed several incubation experiments using fluorescently labeled bacteria (FLB) to measure ingestion by mixotrophic protists under certain treatment conditions.
The issue in analyzing these samples is often that with normal DAPI staining it is very difficult to determine whether a prey particle is ingested by a predator or not. This is mainly because DAPI staining does not show the cell outline clearly. We have made use of primulin once which gives a nice result and stains the membrane quite well, but the overflow of fluorescence from primulin into the channel where I measure my FLB prevents me from using it (intensity of my FLB and carry-over fluorescence from primulin are the same and thus I am unable to distinguish the two).
Now I have also heard that you can use Phalloidin to stain the actin filaments and with it thus also more or less the cell outline.
Does anyone have information or experience with Phalloidin staining and what would be a good way to get a clear distinction between cell boundaries on a filter otherwise? I know of the existence of Vectashield + Phalloidin, but haven't tried it yet. I am curious if anyone has experience with this.
It would be great to hear other experiences or opinions!
Thanks in advance,
Sebastiaan Koppelle,
High Sebastiaan,
I suggest you use F1-43 or FM4-64. They stain membranes, and solely membranes, and are not deleterious for cell survival.
Cheers
Oliv
High Olivier Catrice ,
Thank you for your answer and for thinking along. F1-43 and FM4-64 would possibly be good option in the sense that they only stain the membrane, but the issue with these stains is that they have a very wide excitation emission spectrum which will cause issues when wanting to look into different channels at the same time.
Do you have personal experience with these stains? And also in case you already have a filter? Can you just apply these stains on the filter itself or do you mount it with the stain?
Curious to hear about it,
Thanks,
Sebastiaan
Indeed they have, but if you use GFP, FM4-64 will be OK as it emits in the red part of the spectrum. There is also a FM5-95 but I never used it.
You can read :
https://doi.org/10.1111/j.0022-2720.2004.01348.x which has become a reference paper for these dyes. You will see that there is non problem taking images of FM4-64 and GFP simultaneously.
What kind of filter are you talking about ? Dichroic optical filters ? I don't really understand the question.
I didn't answer your question about phalloidin. Don't use it on live cells, it is a potent inhibitor of actin filament depolymerization. As you are looking at endocytosis, you want to see actin dependent movements, you must not interfere with F-actin. Phalloidin is a good marker for immunofluorescence or staining of quiescent cells.
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