<html><head></head><body><div class="yahoo-style-wrap" style="font-family:times new roman, new york, times, serif;font-size:13px;"><div dir="ltr" data-setdir="false"><font size="3">Dear all,</font></div><div dir="ltr" data-setdir="false"><font size="3"><br></font></div><div dir="ltr" data-setdir="false"><font size="3">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. </font></div><div dir="ltr" data-setdir="false"><font size="3">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.</font></div><div dir="ltr" data-setdir="false"><font size="3">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.</font></div><div dir="ltr" data-setdir="false"><font size="3">Very much appreciate your thoughts and experiences on this. Thank you.</font></div><div dir="ltr" data-setdir="false"><font size="3"><br></font></div><div dir="ltr" data-setdir="false"><font size="3">Many thanks,</font></div><div dir="ltr" data-setdir="false"><font size="3">Qiaohui</font></div></div></body></html>