Hello,
We have a strange phenomenon occurring where RBC are appearing both light and dark in our TEM sections. This occurs with or without staining, in both light and electron microscopy preps. The cells have been plasmodium infected, and processed as follows: 2.5% Glutaraldehyde fixed at least overnight (against our wishes as I recommend a Paraform. Glutaraldehyde mixture for any infected material),1% Osmicated followed by graded ethanol dehydrations and Epon embedding. Sections are 80 nm thick and normally stained with UA followed by Pb.
Any suggestions or references would be highly appreciated.
Sincerely,
Michael Delannoy
I would have to take a look at them, but the content of the infected RBC's change upon growth and maturation of the intracellular Plasmodium due to its metabolism (incl. Hb used by P.sp). CYa.
The iron content will affect the darkness of the RBC and also the maturity. I have been looking at haematopoiesis in developing placenta and it is very apparent there.. some are really pale and these are generally immature.
In some cases when you are working with blood, the centrifugations done in order to obtain the pellet of prefixed and postfixed RBC, could cause a differential staining of the cells as a consequence of the speed of the centrifugation. This is a very common artifact that could be prevented by a low speed pelleting of the cells or by letting the cells down by gravity for a few minutes (15-30 min). I hope that this informationentrifu
After washing the cells and their resuspension in a buffer the fixation should be performed by dropping gently your GA solution until you obtain 2.5 % as wanted. This should really done drop by drop under gentle motion. The next day you can centrifuge, but not more than 1000 x g. I do not know your procedure, but perhaps it can help, if it is really an artifact.
Good luck
We have tried triple staining on these samples (tannic acid followed by uranly acetate then lead citrate), and this helped a lot. Thanks to all for their input.
sincerely,
Michael Delannoy
Hi
is it possible that you are observing infected rbc containing hemazoin?
http://europepmc.org/abstract/med/3529929
cheers
kay
Yes,
That was my first inclination, but the researcher is hard to convince.
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