We are trying to stain live Entamoeba cells with sir Actin( conc- 100nm to 1um) in culture medium. But we are unable to see any staining. Please suggest what may be the problem. We are also using 10um verapamil.
What type of fluorescent microscope are you using? If a normal fluorescent widefield microscope and observation by eye it can be difficult to see for many people as it emits at a max of 673nm (on the limit of our visual range). Try using a good camera on the microscope with the correct filter set or alternatively a confocal microscope with excitation at 633 or 647 nm. It is also possible that Entamoeba cells do not have f-actin? SirActin is very specific for this Actin type.
David G Spiller We are using the olympus FV3000 confocal microscope in the far red range (cy5 filter) as mentioned in the company s protocol. It stains perfectly in fixed cells but not in live cells. Entamoeba do have F actin and stains perfectly with phalloidin.
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