I'm going to view localization of certain fluorescent protein in Enterococuccus. As they tend to form chains, and for analysis I need to use single cells images, I wonder if I can fix in first with PFA and then proceed with microscopy? Are they still considered alive?
Another question is if I want to track the appearance of my protein on the cell surface during the cell growth, may I use agarose pad, or it wouldn't divide on it? Can I use BHI agar instead or it will interfere with imaging due to less-transperent properties?
That's where fixing becomes a problem, once fixed will I be able to track division or not?
Any cell once fixed is very very dead. PFA is a small molecule that rapidly infiltrates cells. Once in, it 'fixes' proteins mainly through the formation of di-sulphide bridges between adjacent cysteine residues. This causes structural anomalies in several metabolic proteins which essentially 'kills' the cells.
Any cell once fixed is very very dead. PFA is a small molecule that rapidly infiltrates cells. Once in, it 'fixes' proteins mainly through the formation of di-sulphide bridges between adjacent cysteine residues. This causes structural anomalies in several metabolic proteins which essentially 'kills' the cells.
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