Dear Alsayed Mohamed,

it would be easy to help you with an answer to your question.

Unfortunately it depends (IMHO) on some parameters of processing you have not provided in your post, such like:

i) (for brain) perhaps also fixation procedure (at least briefly), dehydration, intermedium and

ii) type of resin (embedding)

iii) type of UA-solution: hydrous- & % & acidity, or alcoholic & %, or other (specify), eventually source, storage conditions and age of UO2Ac

iv) assuming LN = Lead Nitrate:

which procedure: e.g. Reynolds, Venable & Coggeshall, other

v) generally, staining by UO2ac is (should be) longer than Lead “citrate” staining, since excess staining by the highly basic lead citrate diminishes / removes bound UO2ac and therefore contrast (has been reported not often in the literature but has been found in published/unpublished abstracts, personal communications as well as own experience).

For e.g. 1% UO2Ac in Methanol 100% + 1-2 drops 100% acetic acid for stabilization and lead citrate (e. g. Venable & Coggeshall, freshly prepared) times could be 15 min UO2 ac. and 0.5 to max. 1.5 min lead citrate.

Unfortunately I cannot serve you with “the best counterstain” recipe you ask for …. I would perform first some test stainings (longer UO2Ac, shorter Lead citrate) and perhaps / for sure you’ll find “routine staining schedules” also for brain sections 50 nm by searching PubMed and Googling. Also, I am confident, there will be other colleagues, more experienced than me, helping you with hard facts about the matter you are asking for.

Best wishes and regards, Wolfgang MUSS, Salzburg

Thanks a lot for your wide explanation, and for that i made my question quite general...Fortunately, i got that protocol (http://www4.utsouthwestern.edu/mcif/pdfs/Staining%20Thin.pdf) today and hopefully working well in addition to optimizing all my procedures according to your valuable suggestions.

Try out Samarium triacetate (e.g. from Sigma), instead of UA. Works as good as UA being not as hazardoous.

Original message from Dr. Muss:

https://www.researchgate.net/post/TEM_Substitute_reagents_for_Uranyl-Acetate_in_positive_and_negative_staining_of_resin_ultrathin_sections_Dear_Colleagues_I_would_like_to_inform_you_of_an_article_which_recently_was_published_in_Journ2

Dear Alsayed Mohamed,

just to add the original references for the Reynolds and Venable&Coggeshall’s Lead Citrate (because they are not mentioned in the mentioned .pdf from web at: http://www4.utsouthwestern.edu/mcif/pdfs/Staining%20Thin.pdf

and another variant of Uranylacetate-staining solution:

Original References:

VENABLE J.H, Coggeshall R. (1965): A Simplified Lead Citrate Stain for Use in Electron Microscopy; J. Cell Biol.(N.Y.) 25, 407-408

REYNOLDS E.S. (1963): The Use of Lead Citrate at High pH as an Electron-opaque Stain in Electron Microscopy; J. Cell Biol.(N.Y.) 17, 208-212

[why [corrected] acetic acid should be added (thanks to Prof. John Bozzola, , Southern Illinois Univ, Carbondale, IL, USA,who pointed me to the following paper): to prolong use of the staining solution without UO4(?) precipitates, cf. eg. Formation of Peroxidic Precipitate in the Radiolysis of Uranyl Nitrate Ketone Solutns, by Jiri Teply and Vaclav Stulik, in Nature Vol.200, No 4907,Nov.16th (1963): what is valid for Uranylnitrate may be true also for UO2-Acetate]:

0.5 g uranyl acetate-dihydrate carefully are weighed into 50 ml of 100% methanol (cave: methanol will be / is hygroscopic), stirred at least for 2 hrs with a magnetic bar (e.g. shielding from light by Al-foil is recommended). After complete dissolution of UO2Ac-powder, add 2-5 drops of (100%=glacial) acetic acid to the solution, let stir for some minute and suck into absolutely clean (means: wash&spill at least 2-3 times with 100% methanol to get rid of e.g. silicone oil inside) single-use syringes = 2x 25 ml, mount Swinex or Millipore microfilter (0.2 or 0.45 µm pore size), press gently until UO2-Ac-solution has moistened whole filter and shut opening of microfilter with a respective tight plastic plug. You can use such a working solution for as long as 3-4 weeks (light shielding and tight fastening of the plug provided, until eventually a precipitate will form. You can use the solution as long as the precipitate is consisting of only few crystals and as long as the solution retains its YELLOW color).

Use for epoxide (e.g. Epon & substitutes) resin sections [90-50 nm and thinner]:

standard incubation @RT in a black glass through (embryo dish) with black cover-plate for 15 min, jet-washing with Aqua bidest., short blotting with the edge of a filter paper (including the angle between tweezer handles), followed by 1.5 to 3 min on a drop of lead citrate (e.g. Venable & Coggeshall 1965).

By the way: cf. also my post dated Dec 7, 2011

at see above, thanks to Yaroslav Tsytsyura to cite this one...(:-)):

Hope to have been of help anyway, best wishes and good luck,

Wolfgang