Immunofluorescence staining is applied to cells either as in-vitro cell culture or in vivo animal models, and localisation of infectious agents is determined via the immunofluorescence signals. How sure are you that these signals are intracellular or extracellular? And what modifications would you do to improve the results of the localisation study (such as adding a procedure to lyse the cells to bind the antibody intracellularly)?
Hi, simply by keeping a control, for example here, one set of samples 1) adding a procedure to lyse/ permeabilize the cells to bind the antibody intracellularly and another sample set with only PBS or physiological saline and compare the result.
Positive control may be also applied to know if binding is specific
In particular, as I can guess from your question: when your "signal" is immunolabeled bacteria and you are interested in whether they are inracellular or simply attached to the surface of cultured cells: in addition to immunofluorescence labeling you can apply microbiological culture methods - first kill the extracellular bacteria with antibiotics, then lyse the cells and plate on agar.
In my experience with immunofluorescence I would suggest what Chandra said. So, if you are unsure of where to find the antigen (infectious agent in your case), prepare two sets of samples for each experiments.
a) Fix cells in 2% paraformaldehyde for 10 min at RT or for 30 min on ice.
b) Fix and permeabilize cells with cold methanol for 5 min. This way antibodies should be able to get inside the cells.
This worked well for me in colorectal cancer cells. I was able to distinguish perfectly intracellular from extracellular staining, although I guess depending on the antigen it can be tricky.
I suppose that this protocol does not work for bacteria nor for plant cells.
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