Hi everyone,
I met a problem recently and really need your advise. In the last two rounds of sample embedding for TEM with resin, we found uranyl acetate precipitation right away after sectioning (before staining). This has never happened before. We used brand new uranyl acetate but the problem was still there.
My samples are Agro-infiltrated tobacco leaves and embedding started at 4th day after infiltration. Photos of the precipitation are attached.
Thanks a lot in advance!
If it is residual PO4 / phosphate salts, as Nicolas Barois anticipates, this may be due to poor / inconsequent processing
( i.e. using PO4-buffer for washing and precipitating during dehydration by a first ethanol concentration higher than 70% ,
or - more likely - NOT using the Maleate buffer sequence AFTER use of PO4-buffer - before UO2-acetate incubation prior to further dehydration and embedding).
As Vladimir Dusevich points out the described and imaged precipitations seem to be quite selective and very big and I am not convinced about their nature being UO2-crystals ONLY .
It would help a lot to get the whole protocol you used for the specific samples you showed in your images - step by step until embedding.....then there - for sure- are following serious explanations. Best, W.H.M.
Its difficult to give an advice because your pictures are rather unusual.Your precipitates are very selective in their placement and crystals are so big they cause tears/holes in your sections. Not our typical UA precipitation. Of course you can try to check with EDS (TEM or SEM) if it is really UA, but you had good sections previously. It tells that this time just some mistake had place. So, instead of spending time on tracing cause of this mistake you can just prepare new specimens with brand new chemicals and enjoy new good sections.
By the way, I prefer post embedding staining just because mistakes made on preembedding stage are more costly.
Hello,
Uranyl acetate precipitates in presence of phosphate salts. So maybe at first, it is to check you are well washing your sample with water before using UA and not with PBS for example. Mistake between bottles or by new experimenter can happen (I recall a new experimenter killing all his cell cultures by washing them with 10X PBS.)
Precipitate looks only inside your sample, between the two sides of the leaf.
It may be related to the permeability of plant tissues. Phosphate salts may not have been washed away.
So if it is not due to phosphate salts, i don't see another clue yet.
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