We want to know localization of Calcium in the nucleus or nucleolus or chromatin of HeLa cells and also looking for staining but after staining in the transmission electron microscopy we see lot of precipitate.
I want to ask if someone working in Calcium detection, Calcium signaling or effect of Ca2+ in nucleus please let me know.
Thanks
Which electron microscope? TEM, SEM?
Whose nucleus?
Do you use EDS?
What kind of staining have you used?
In my knowledge, there are not cytochemical methods to demonstrate directly calcium in the nucleus. It is possible to perform immunocytochemical reactions with markers of proteins binding with calcium such as calbindin or PMCA.
Graziella is completely right: Calcium ions cannot be directly visualized, wether in the nucleus or somewhere else. On the other hand, employing the right labels it is possible to determine intracellular calcium (even quantitatively), e.g. with fluorescent labels (FURA, OregonGreen etc) and a fluorescence microscope. An electron microscope (TEM, SEM) will be absolutely useless for this purpose. In theory you may look for a calcium chelator, which forms electron-dense aggregates, which may be visualized in your TEM. This is certainly not the quantitative approach you are looking for.
Actually, electron microscope (STEM, SEM) with EDS could be the best way to look for local variations in Ca concentration.
maybe potassium pyroantimoniate-osmium method? see:
1) Appleton J., Morris D. C. (1979) The use of potassium pyroantimoniate-osmium method as a means of identifying and localizing calcium at the ultrastructural level in the cells of calcifying systems. J. Histochem. Cytochem. 27:676–680.
2) Lu et al., (1998) Ca2+ content and expression of an acidocalcisomal calcium pump are elevated in intracellular forms of Trypanosoma cruzi. Mol Cell Biol. 18(4):2309-23.
at the first thanks to all for your answers.
@ Dear Vladimir: We are working with TEM and HeLa cells. (I Edited my question)
@ Dear Graziella: we are looking to Free Calcium in nucleus and in general we can obtain result with calcium detector with fluorescent labels as JUST GENIUS wrote. but we are looking for method to study with EM not fluorescent so this methods are not useful for us.
@ Dear Just Genius: You right this methods are useless for EM and this is the point that we are searching a method to study calcium with TEM.
@ Dear David: these articles are really good references but I don't know that we can apply these methods to HeLa cells and also studying free calcium in nucleus with this methods on the normal conditions or no. because they studied about calcifying system and also intracellular parasite and some special cells and conditions.
Have you successfully detection the free calcium concentration around your Hela cells?
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