Hi guys,
I’m experiencing some very small black spots in my electron micrographs. So far I noticed them in particular in tissues/cells that contain glycogen granules…at least this is what seems to me. I was wondering if those smal spots could be due from a kind of glycogen “degradation” during the embedding procedure.
Or maybe they are due to something else?
The pictures I attached are from cultured fibroblasts
Many thanks to those that would like to give me some suggestion
Francesco
looks like lead precipitate from the section staining procedure to me
But it is strange, usually I never have this spots in different samples
As always: it ('interpretation') depends Not "Sergeant Pepper", but 'Salt & Pepper'*), but is is only a guess ...unless you might / would like to provide your specimen processing method (i.e., specimen, treatment, solutions used,...etc.; esp. from first fixation until last step of dehydration ...)
*) well known for long -esp. when using PO4-buffered fix. and/or washing buffer(s)....
Thanks for more detailed information....
Before commenting, I would like to ask what was the buffer you used during the fixation and washing steps?
Usually I fix cells in GA 2.5% in 0,1M sodium cacodylate buffer pH 7.4, then 3 washes with buffer, post fixation in 1% OsO4 in the same buffer, 3 washes, deydratation from 25% EtOh to 3X 100% EtOh,
Good, as a first guess - phosphate precipitates may be formed if you have phosphates in the buffers, eg, PBS, but you use CB. I agree with you that the rosettes you identify as glycogen are this. But in my opinion the tiny precipitates in your samples might be some artefact due to counterstaining, most likely - lead containing precipitates. In my lab, to avoid this, during counterstaining which we used to put some NaOH crystals in a petri dish near the drops with the counterstains, all these covered under the same box. Sometimes helps.
I use NaOH crystals too during lead counterstaining.
Many thanks!!!
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