Hi Martin,
if you want to see how the Nosema infection affects the intracellular distibution of Ca++, you really need a sensitive fluorescent stain applicable to a living cell. It is a tricky thing and in many cases needs an in vitro system, which for a microsporidium is not an easy task as well. See for instance http://www.invitrogen.com/etc/medialib/en/filelibrary/Support/BioProbes/BioProbes-63.Par.73164.File.dat/BioProbes_63-Calcium-Imaging.pdf
Take a look at mito stains there considering the interactions of an intracellular microsporidium and the host cell mitos.
Many years ago, I used to work in Prof. V.V. Glupov's lab (Novosibirsk, Russia) and they used chlortetracycline (aureomycin) to stain haemocytes for Ca++ distribution during insect immune reaction. It was methologically as the free haemocytes were isolated as suspended cells from naive and challenged insects. For honey bee ventriculus, it will not be possible..... BUT you won't see anything with a histo stain, that's I'm sure!
If you can confocal isolated tissue of honey bee you can stain'em with some fluorescent dyes and possibly see the parasites within the host cell cytoplasm. We did such things but usually with immunolabels. OR is it somwthing else that you're looking for??
Hi Yuri,
Thanks for getting back to me. We've been using Alizarin Red A, Biebrich's solution, two fluorescent green stains, and H&E to stain healthy and infected midgut tissue. Kohler (1920), Frenzel (1886), and Hertig (1923) postulate that Ca2+ (possibly as an oxalate or carbonate) accumulates in the epithelial cells of the ventriculus where it acts as a buffer against the digestive acids.
We've developed a staining technique that clearly shows what appears to be sequestered refractive bodies (Ca2+??); however, without a road map to show us what we're looking at, we can only surmise that what we have is Ca2+.
There's no problem staining the spores in the ventriculus. I''ve been doing that for a couple of years now using Trypan Blue and Calcofuor White and viewing under FM. I'm intrigued by your work with chlortetracycline (aureomycin) to stain haemocytes for Ca++ distribution during insect immune reaction. I've been looking over Gilliam's early work on honey bee hemocytes. I might try using aureomycin to stain the hemocytes for signs of sporonts or meronts. Beyond that, I imagine the only other option I have is to dissolve the midgut tissue in nitric acid and then measure the Ca2+ levels that were in the ventriculus (assuming that what I'm seeing histologically is Ca2+). I'll keep you posted.
As you mentioned, our next step is to view the cells using confocal microscopy and TEM.
Thanks again.
Ok, I got it. You are looking for granules of Calcium salts (I thought it is phosphate) in infected vs unifected cells. I am afraid the difference may be neglible in terms of quantity; it may be just redistribution of ions which all will be equalized after you dissolve the tissue in an acid. However, if you see that abundance of granules is very different in infected vs uninfected cells (or bees), you might be lucky with quantification.
good luck, then
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