<html><head><meta http-equiv="Content-Type" content="text/html; charset=UTF-8"></head><body dir="auto"><div><br></div><div style="font-size:100%;color:#000000" dir="auto"></div><div style="font-size:100%;color:#000000" dir="auto"><!-- originalMessage --><div>Hi <span style="font-size: 100%;">Qiaohui Yang,</span></div><div><span style="font-size: 100%;">There are two solutions to your problem,</span></div><div><span style="font-size: 100%;">One is to make the freeze-substition mixture without water (so for 5ml end volume dissolve the osmium and uranyl acetate in 4,75 ml acetone), cool it down to -90°C and then pipette in the water (this needs to be done quickly otherwise the water freezes in the pipette tip). Then vortex the solution until all visible ice crystals have dissolved and cool down the mixture again. </span></div><div><span style="font-size: 100%;">The othet solution is to use a stock solution of 20% uranyl acetate dissolved in dry methanol.</span></div><div>(This was also done in tyhe walther & ziegler paper on the addition of water in freeze substitution).</div><div>In my hands with using the first method 1% of water is enough to enhance membrane contrast, going to 3% enhances the visibility of glycogen and not contrast is gained from going to 5%. The small ammount of methanol added in method 2 allows for more water to be dissolved in the substitution mix at lower temperatures, so more water needs to be added to get the same contrast enhancement effect.</div><div><br></div><div>With best regards,</div><div><br></div><div>Rob Mesman</div><div><br></div><div>Dr. Rob Mesman<br>Post-Doc<br>Department of Microbiology<br>Faculty of Science<br>Radboud University Nijmegen<br>Heyendaalseweg 135<br>NL-6525 AJ Nijmegen<br>The Netherlands</div></div><br dir="auto">Message: 2<br dir="auto">Date: Thu, 19 Dec 2019 08:39:29 +0000 (UTC)<br dir="auto">From: Qiaohui Yang <qiaohui.yang@ymail.com><br dir="auto">To: 3dem@ncmir.ucsd.edu<br dir="auto">Subject: [3dem] Precipitation observed after making freeze<br dir="auto"> substitution cocktail containing 5% water<br dir="auto">Message-ID: <1061929162.800832.1576744769397@mail.yahoo.com><br dir="auto">Content-Type: text/plain; charset="utf-8"<br dir="auto"><br dir="auto"><br dir="auto">Hi everyone!<br dir="auto">We recently tried to make freeze substitution cocktailconsisting of 1% osmium tetroxide, 0.1% uranyl acetate and 5% water but observedthat the mixture turned slightly cloudy within 1-2 minutes. We tried to see if the precipitation would occur in the absence of osmium tetroxide, and we observed some cloudiness too after mixing uranyl acetate solution in water with pure acetone.<br dir="auto">Just wondering if anyone has encountered similar problem andif so, would appreciate some tips on how to overcome this problem.<br dir="auto"><br dir="auto">Thank you!<br dir="auto"><br dir="auto">?<br dir="auto"><br dir="auto">Best regards,<br dir="auto"><br dir="auto">Qiaohui<br dir="auto"><br dir="auto">-------------- next part --------------<br dir="auto">An HTML attachment was scrubbed...<br dir="auto">URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20191219/aa9c3a85/attachment.html><br dir="auto"><br dir="auto">------------------------------<br dir="auto"><br dir="auto">Subject: Digest Footer<br dir="auto"><br dir="auto">_______________________________________________<br dir="auto">3dem mailing list<br dir="auto">3dem@ncmir.ucsd.edu<br dir="auto">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem<br dir="auto"><br dir="auto"><br dir="auto">------------------------------<br dir="auto"><br dir="auto">End of 3dem Digest, Vol 148, Issue 22<br dir="auto">*************************************<br dir="auto"></body></html>