I am trying for a long time to localize loosely bound calcium ions in Drosophila tissues using pyroantimonate precipitation for TEM analysis, however every time I do not have any precipitates. I do not know if it is caused by wrong concentration of PPA or I do something wrong with pH of buffers. Is any one could give me FULL protocol for PPA precipitation in animal tissue including preparation of PPA solution? Currently I am trying do this using potassium phosphate buffer, pH 7.4, I add PPA to fixative. I would be greatful for any suggestions.
Dear Przemysław Zakrzewski, would you mind to inform a bit more detailed
i) about the "drosophila tissues " you examine
ii) which original recipe you have been using and
iii) how you prepare your buffers or other solutions.
It might be of benefit to come to an honest conclusion how one can help (or if someone might be of help anyway )... TIA, regards, WM
Dissolve 2 g of potassium pyroantimonate in 85 mL of hot water. Cool as fast as you can (on a crushed iced bath) and add 10 mL of a KOH solution. Incubate at room temperature for 24 h. Filter and add water to 100 mL.
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