[3dem] 1. Re: Precipitation observed after making freeze substitution cocktail containing 5% water (Qiaohui Yang)

Fri Jan 17 06:59:06 PST 2020

Hi there,

Any time I had a precipitate (unrelated to this particular FS cocktail you are asking about), I had problems with crystals and whatnot in the final sections. You definitely want to avoid the precipitate.

I haven’t made this particular FS cocktail in a little over 5 years (new job with way less FS). At any rate, I recall doing nothing special. I just used our regular 2% UA in dH20 that I used for post-staining grids. I also never worried about if it was fresh 2% UA. It was convenient because the FS recipe would be 0.5 g OsO4 crystal, 2.5 ml of 2% aqueous UA and 47.5 ml anhydrous acetone. I would just mix it, aliquot it at room temp, and freeze it. I just didn’t have a precipitate problem. And, with dealing with OsO4 crystals, I would always wash the vial with anhydrous acetone and use a glass bottle that the interior was washed with anhydrous acetone—this is important b/c any contaminants will cause the osmium to react immediately. Then, I would put the vial in the bottle, put the cap on the bottle, and shake it until the vial broke. Then, I would add the 2% UA and acetone to the bottle, make sure the OsO4 dissolved, and aliquot from there (using very clean pipettes/cryo-vials). Again, I never had a precipitate problem. All reagents were from EMS.  I don’t know if it matters if you add the water component first or the acetone component first. I would imagine I didn’t care too much and just added what was most convenient first.

Incidentally, the only time I ever had a precipitate problem was when I tried to figure out a reduced osmium for FS. The K4Fe(CN)6 would always precipitate out so I abandoned it.

Here’s how I make 2% UA:
Dump old UA into waste container
Boil dH2O
Add bdH2O and about 1ml glacial acetic acid to UA container and sonicate to clean, dump into waste container
Add 5g UA to 250ml bdH2O
Sonicate for about 10min
Wrap in foil
Add 2-3 drops glacial acetic acid

I hope that helps,
Cindi L. Schwartz

Electron Microscopist
Rocky Mountain Labs/NIAID/NIH
903 South 4th Street
Hamilton, MT  59840
406-363-9228
Cindi.Schwartz at NIH.gov<mailto:Cindi.Schwartz at NIH.gov>
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From: Qiaohui Yang <qiaohui.yang at ymail.com>
Sent: Friday, January 17, 2020 2:43 AM
To: 3dem at ncmir.ucsd.edu
Subject: [3dem] 1. Re: Precipitation observed after making freeze substitution cocktail containing 5% water (Qiaohui Yang)

Dear all,

thank you so much for your valuable inputs following my post on this issue with precipitation observed while making freeze substitution cocktail with 5% water.
To place this topic back into context, I was actually trying to follow the freeze substitution protocol as described in Buser et al. 2008 which describes the positive effects of adding some water to polar solvents such as acetone for improved ultrastructural preservation. When we first attempted this method, we observed precipitation which developed within several minutes after mixing osmium tetroxide in acetone with uranyl acetate dissolved in CO2free water at room temperature to achieve a final FS mixture consisting of 1%OsO4, 0.1%UA with 5% water.
Thanks to the advice given by Christopher who suggested to cool the mixtures down, we found that the rate of precipitation was greatly reduced when the mixing was carried out at around -20degrees celsius before we immediately froze the FS cocktail in liquid nitrogen. Nevertheless, we are still wondering how this precipitation will impact biological structures during the actual freeze substitution process since the cocktail will still be warmed up from -90degrees to 0 degrees eventually anyway.
Very much appreciate your thoughts and experiences on this. Thank you.

Many thanks,
Qiaohui
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