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<div class=Section1>

<p class=MsoNormal><span class=SpellE><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;font-family:Arial;color:navy'>Hilal</span></font></span><font
size=2 color=navy face=Arial><span style='font-size:10.0pt;font-family:Arial;
color:navy'>:<o:p></o:p></span></font></p>

<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p>&nbsp;</o:p></span></font></p>

<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>I assume that the reason you want to
remove the cell wall is for osmium fixation (cell wall has to be removed in
order for osmium getting into the cell). There is an alternative for fixing the
yeast cells using </span></font><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;mso-bidi-font-size:9.5pt;font-family:Arial;mso-bidi-font-family:
AdvMinionNormal_Rm;color:navy'>potassium permanganate, instead of </span></font><font
size=2 color=navy face=Arial><span style='font-size:10.0pt;font-family:Arial;
color:navy'>osmium. Permanganate preserves membrane structure very well (see
references below). Here is the protocol &#8211; <o:p></o:p></span></font></p>

<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p>&nbsp;</o:p></span></font></p>

<ol style='margin-top:0in' start=1 type=1>
 <li class=MsoNormal style='color:navy;mso-list:l1 level1 lfo1;tab-stops:list .5in;
     mso-layout-grid-align:none;text-autospace:none'><font size=2 color=navy
     face=Arial><span style='font-size:10.0pt;font-family:Arial'>Harvested
     yeast cells <span class=GramE>are fixed</span> first with </span></font><font
     size=2 face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:9.5pt;
     font-family:Arial;mso-bidi-font-family:AdvMinionNormal_Rm'>2.5% (v/v) glutaraldehyde
     in PBS for 40 min at room temperature.<o:p></o:p></span></font></li>
 <li class=MsoNormal style='color:navy;mso-list:l1 level1 lfo1;tab-stops:list .5in;
     mso-layout-grid-align:none;text-autospace:none'><font size=2 color=navy
     face=Arial><span style='font-size:10.0pt;font-family:Arial'>Rinse with PBS
     3X<o:p></o:p></span></font></li>
 <li class=MsoNormal style='color:navy;mso-list:l1 level1 lfo1;tab-stops:list .5in;
     mso-layout-grid-align:none;text-autospace:none'><font size=2 color=navy
     face=Arial><span style='font-size:10.0pt;font-family:Arial'>Cells <span
     class=GramE>are further fixed</span> with </span></font><font size=2
     face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:9.5pt;
     font-family:Arial;mso-bidi-font-family:AdvMinionNormal_Rm'>2% potassium
     permanganate in water for 1 h at room temperature.</span></font><font
     size=2 face=Arial><span style='font-size:10.0pt;font-family:Arial'><o:p></o:p></span></font></li>
 <li class=MsoNormal style='color:navy;mso-list:l1 level1 lfo1;tab-stops:list .5in;
     mso-layout-grid-align:none;text-autospace:none'><font size=2 color=navy
     face=Arial><span style='font-size:10.0pt;font-family:Arial'>Rinse with
     water <o:p></o:p></span></font></li>
 <li class=MsoNormal style='color:navy;mso-list:l1 level1 lfo1;tab-stops:list .5in;
     mso-layout-grid-align:none;text-autospace:none'><font size=2 color=navy
     face=Arial><span style='font-size:10.0pt;font-family:Arial'>Dehydration as
     usual <o:p></o:p></span></font></li>
 <li class=MsoNormal style='color:navy;mso-list:l1 level1 lfo1;tab-stops:list .5in;
     mso-layout-grid-align:none;text-autospace:none'><font size=2 color=navy
     face=Arial><span style='font-size:10.0pt;font-family:Arial'>&#8230;..<o:p></o:p></span></font></li>
</ol>

<p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><font
size=2 color=navy face=Arial><span style='font-size:10.0pt;font-family:Arial;
color:navy'><o:p>&nbsp;</o:p></span></font></p>

<p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><font
size=2 color=navy face=Arial><span style='font-size:10.0pt;font-family:Arial;
color:navy'>The potassium permanganate will stain cells black, as osmium, so
that you can easily find cells for section.<o:p></o:p></span></font></p>

<p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><font
size=2 color=navy face=Arial><span style='font-size:10.0pt;font-family:Arial;
color:navy'><o:p>&nbsp;</o:p></span></font></p>

<p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><font
size=2 color=navy face=Arial><span style='font-size:10.0pt;font-family:Arial;
color:navy'>If you DO need to remove the cell wall for <span class=GramE>reasons</span>
I am unaware of. Here is the protocol<o:p></o:p></span></font></p>

<ol style='margin-top:0in' start=1 type=1>
 <li class=MsoNormal style='color:navy;mso-list:l0 level1 lfo2;tab-stops:list .5in;
     mso-layout-grid-align:none;text-autospace:none'><font size=2 color=navy
     face=Arial><span style='font-size:10.0pt;font-family:Arial'>Harvested
     yeast cells <span class=GramE>are fixed</span> first with </span></font><font
     size=2 face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:9.5pt;
     font-family:Arial;mso-bidi-font-family:AdvMinionNormal_Rm'>2.5% (v/v) glutaraldehyde
     in PBS for 40 min at room temperature.<o:p></o:p></span></font></li>
 <li class=MsoNormal style='color:navy;mso-list:l0 level1 lfo2;tab-stops:list .5in;
     mso-layout-grid-align:none;text-autospace:none'><font size=2 color=navy
     face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:9.5pt;
     font-family:Arial;mso-bidi-font-family:AdvMinionNormal_Rm'>Rinse</span></font><font
     size=2 face=Arial><span style='font-size:10.0pt;font-family:Arial'><o:p></o:p></span></font></li>
 <li class=MsoNormal style='color:navy;mso-list:l0 level1 lfo2;tab-stops:list .5in;
     mso-layout-grid-align:none;text-autospace:none'><font size=2 color=navy
     face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:9.5pt;
     font-family:Arial;mso-bidi-font-family:AdvMinionNormal_Rm'>Cells are
     digested with <span class=SpellE>Zymolyase</span> 100T (0.3mg/L, final
     concentration) for 10 &#8211; 30 min to remove the cell wall.</span></font><font
     size=2 face=Arial><span style='font-size:10.0pt;font-family:Arial'><o:p></o:p></span></font></li>
 <li class=MsoNormal style='color:navy;mso-list:l0 level1 lfo2;tab-stops:list .5in;
     mso-layout-grid-align:none;text-autospace:none'><font size=2 color=navy
     face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:9.5pt;
     font-family:Arial;mso-bidi-font-family:AdvMinionNormal_Rm'>Cells <span
     class=GramE>are further fixed</span> with 1% osmium in water or buffer for
     1 hour at room temperature.</span></font><font size=2 face=Arial><span
     style='font-size:10.0pt;font-family:Arial'><o:p></o:p></span></font></li>
 <li class=MsoNormal style='color:navy;mso-list:l0 level1 lfo2;tab-stops:list .5in;
     mso-layout-grid-align:none;text-autospace:none'><font size=2 color=navy
     face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:9.5pt;
     font-family:Arial;mso-bidi-font-family:AdvMinionNormal_Rm'>Rinse</span></font><font
     size=2 face=Arial><span style='font-size:10.0pt;font-family:Arial'><o:p></o:p></span></font></li>
 <li class=MsoNormal style='color:navy;mso-list:l0 level1 lfo2;tab-stops:list .5in;
     mso-layout-grid-align:none;text-autospace:none'><font size=2 color=navy
     face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:9.5pt;
     font-family:Arial;mso-bidi-font-family:AdvMinionNormal_Rm'>Dehydration</span></font><font
     size=2 face=Arial><span style='font-size:10.0pt;font-family:Arial'><o:p></o:p></span></font></li>
 <li class=MsoNormal style='color:navy;mso-list:l0 level1 lfo2;tab-stops:list .5in;
     mso-layout-grid-align:none;text-autospace:none'><font size=2 color=navy
     face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:9.5pt;
     font-family:Arial;mso-bidi-font-family:AdvMinionNormal_Rm'>&#8230;.</span></font><font
     size=2 face=Arial><span style='font-size:10.0pt;font-family:Arial'><o:p></o:p></span></font></li>
</ol>

<p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><font
size=2 color=navy face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:
9.5pt;font-family:Arial;mso-bidi-font-family:AdvMinionNormal_Rm;color:navy'><o:p>&nbsp;</o:p></span></font></p>

<p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><font
size=2 color=navy face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:
9.5pt;font-family:Arial;mso-bidi-font-family:AdvMinionNormal_Rm;color:navy'>The
time on step 3 is critical for your success (i.e. remove the cell wall without
damaging the cell). For the purpose of fixation (of osmium), you really <span
class=GramE>don&#8217;t</span> need remove 100% of the cell wall. Partially remove
it would be best. When you do the digestion, for every 10 min or so, take some
cells and check under a compound microscope. Once you see the cell shaped
changed (such as from football shape to round), it is time to stop the digestion.
Otherwise, it might be too much (even though it may not be enough for making spheroplast).<o:p></o:p></span></font></p>

<p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><font
size=2 color=navy face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:
9.5pt;font-family:Arial;mso-bidi-font-family:AdvMinionNormal_Rm;color:navy'><o:p>&nbsp;</o:p></span></font></p>

<div>

<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Hope this helps.<o:p></o:p></span></font></p>

<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p>&nbsp;</o:p></span></font></p>

<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>Zhaojie <o:p></o:p></span></font></p>

<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p>&nbsp;</o:p></span></font></p>

<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>References:<o:p></o:p></span></font></p>

<p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><span
class=GramE><font size=2 color=navy face=Arial><span style='font-size:10.0pt;
mso-bidi-font-size:9.0pt;font-family:Arial;mso-bidi-font-family:NewCenturySchlbk-Roman;
color:navy'>ROBIN WRIGHT (2000) </span></font><font size=2 color=navy
face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:18.0pt;font-family:
Arial;mso-bidi-font-family:HelveticaNeue-Roman;color:navy'>Transmission
Electron Microscopy of Yeast.</span></font></span><font size=2 color=navy
face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:18.0pt;font-family:
Arial;mso-bidi-font-family:HelveticaNeue-Roman;color:navy'> </span></font><font
size=2 color=navy face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:
8.0pt;font-family:Arial;mso-bidi-font-family:NewCenturySchlbk-Roman;color:navy'>MICROSCOPY
RESEARCH AND TECHNIQUE 51:496&#8211;510</span></font><font size=2 color=navy
face=Arial><span style='font-size:10.0pt;font-family:Arial;mso-bidi-font-family:
NewCenturySchlbk-Roman;color:navy'><o:p></o:p></span></font></p>

<p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><font
size=2 color=navy face=Arial><span style='font-size:10.0pt;font-family:Arial;
mso-bidi-font-family:AdvFrutiger-R;color:navy'>Hui Yang, <st1:PersonName w:st="on">Qun
 Ren</st1:PersonName> &amp; Zhaojie Zhang (2006) </span></font><span
class=SpellE><font size=2 color=navy face=Arial><span style='font-size:10.0pt;
mso-bidi-font-size:15.0pt;font-family:Arial;mso-bidi-font-family:AdvT282;
color:navy'>Chromosomeor</span></font></span><font size=2 color=navy
face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:15.0pt;font-family:
Arial;mso-bidi-font-family:AdvT282;color:navy'> chromatin condensation leads <span
class=SpellE>tomeiosis</span> or apoptosis in stationary yeast (</span></font><font
size=2 color=navy face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:
15.5pt;font-family:Arial;mso-bidi-font-family:AdvT282;color:navy'>Saccharomyces
<span class=GramE>cerevisiae <span style='mso-bidi-font-size:15.0pt'>)</span></span></span></font><font
size=2 color=navy face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:
15.0pt;font-family:Arial;mso-bidi-font-family:AdvT282;color:navy'> cells </span></font><font
size=2 color=navy face=Arial><span style='font-size:10.0pt;mso-bidi-font-size:
6.5pt;font-family:Arial;mso-bidi-font-family:AdvFrutiger-L;color:navy'>FEMS
Yeast Res </span></font><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;mso-bidi-font-size:6.5pt;font-family:Arial;mso-bidi-font-family:
AdvFrutiger-B;color:navy'>xx </span></font><font size=2 color=navy face=Arial><span
style='font-size:10.0pt;mso-bidi-font-size:6.5pt;font-family:Arial;mso-bidi-font-family:
AdvFrutiger-L;color:navy'>(2006) 1&#8211;10</span></font><font size=2
color=navy face=Arial><span style='font-size:10.0pt;font-family:Arial;
mso-bidi-font-family:AdvFrutiger-L;color:navy'><o:p></o:p></span></font></p>

<p class=MsoNormal style='mso-layout-grid-align:none;text-autospace:none'><font
size=2 face=AdvT282><span style='font-size:10.0pt;font-family:AdvT282;
mso-bidi-font-family:AdvT282'><o:p>&nbsp;</o:p></span></font></p>

<p class=MsoAutoSig><b style='mso-bidi-font-weight:normal'><font size=2
face="Wendy Medium"><span style='font-size:11.0pt;font-family:"Wendy Medium";
font-weight:bold;mso-bidi-font-weight:normal;mso-no-proof:yes'>Zhaojie Zhang, </span></font></b><font
size=2 face=Arial><span style='font-size:11.0pt;font-family:Arial;mso-no-proof:
yes'>Ph.D.<o:p></o:p></span></font></p>

<p class=MsoAutoSig><font size=2 face=Arial><span style='font-size:11.0pt;
font-family:Arial;mso-no-proof:yes'>Director, Microscopy Core Facility<o:p></o:p></span></font></p>

<p class=MsoAutoSig><st1:place w:st="on"><st1:PlaceType w:st="on"><font size=2
  face=Arial><span style='font-size:11.0pt;font-family:Arial;mso-no-proof:yes'>University</span></font></st1:PlaceType><font
 size=2 face=Arial><span style='font-size:11.0pt;font-family:Arial;mso-no-proof:
 yes'> of <st1:PlaceName w:st="on">Wyoming</st1:PlaceName></span></font></st1:place><font
size=2 face=Arial><span style='font-size:11.0pt;font-family:Arial;mso-no-proof:
yes'><o:p></o:p></span></font></p>

<p class=MsoAutoSig><st1:place w:st="on"><st1:City w:st="on"><font size=2
  face=Arial><span style='font-size:11.0pt;font-family:Arial;mso-no-proof:yes'>Laramie</span></font></st1:City><font
 size=2 face=Arial><span style='font-size:11.0pt;font-family:Arial;mso-no-proof:
 yes'>, <st1:State w:st="on">WY</st1:State> <st1:PostalCode w:st="on">82071</st1:PostalCode></span></font></st1:place><font
size=2 face=Arial><span style='font-size:11.0pt;font-family:Arial;mso-no-proof:
yes'><o:p></o:p></span></font></p>

<p class=MsoAutoSig><font size=2 face=Arial><span style='font-size:11.0pt;
font-family:Arial;mso-no-proof:yes'><a href="zzhang@uwyo.edu">zzhang@uwyo.edu</a><o:p></o:p></span></font></p>

<p class=MsoAutoSig><font size=2 face=Arial><span style='font-size:11.0pt;
font-family:Arial;mso-no-proof:yes'><a href="http://www.uwyo.edu/microscopy">http://www.uwyo.edu/microscopy</a></span></font><font
face=Arial><span style='font-family:Arial;mso-no-proof:yes'><o:p></o:p></span></font></p>

<p class=MsoAutoSig><font size=3 face=Arial><span style='font-size:12.0pt;
font-family:Arial;mso-no-proof:yes'><o:p>&nbsp;</o:p></span></font></p>

<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'><o:p>&nbsp;</o:p></span></font></p>

<div class=MsoNormal align=center style='text-align:center'><font size=3
face="Times New Roman"><span style='font-size:12.0pt'>

<hr size=3 width="100%" align=center tabindex=-1>

</span></font></div>

<p class=MsoNormal><b><font size=2 face=Tahoma><span style='font-size:10.0pt;
font-family:Tahoma;font-weight:bold'>From:</span></font></b><font size=2
face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma'> Hilal Ilarslan
[mailto:ihilal@iastate.edu] <br>
<b><span style='font-weight:bold'>Sent:</span></b> Wednesday, August 30, 2006
5:43 AM<br>
<b><span style='font-weight:bold'>To:</span></b> yeast@magpie.bio.indiana.edu<br>
<b><span style='font-weight:bold'>Subject:</span></b> [Yeast] TEM procedure
using cell wall removal!</span></font><o:p></o:p></p>

</div>

<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'><o:p>&nbsp;</o:p></span></font></p>

<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'>Hi,<br>
<br>
I am working on Saccharomyces cerevisiae. I would like to get help about TEM
technique with cell wall removal without giving any damage to Yeast cell. Is
any one wiilling to give me protocol which I can be succesfull on that. Thanks,
Hilal<br style='mso-special-character:line-break'>
<![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'>
<![endif]><o:p></o:p></span></font></p>

<x-sigsep>

<p style='margin-bottom:12.0pt'></x-sigsep><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'>&nbsp;<o:p></o:p></span></font></p>

<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'>Hilal Ilarslan&nbsp; Ph. D. <o:p></o:p></span></font></p>

<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'>Depart. of Genetics, Development and Cell Biology <o:p></o:p></span></font></p>

<p class=MsoNormal style='margin-left:.5in'><st1:place w:st="on"><st1:PlaceName
 w:st="on"><font size=3 face="Times New Roman"><span style='font-size:12.0pt'>Iowa</span></font></st1:PlaceName>
 <st1:PlaceType w:st="on">State</st1:PlaceType> <st1:PlaceType w:st="on">University</st1:PlaceType></st1:place>
<o:p></o:p></p>

<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'>Room 1 Bessey Hall <o:p></o:p></span></font></p>

<p class=MsoNormal style='margin-left:.5in'><st1:place w:st="on"><st1:City
 w:st="on"><font size=3 face="Times New Roman"><span style='font-size:12.0pt'>Ames</span></font></st1:City>,
 <st1:State w:st="on">Iowa</st1:State>&nbsp; <st1:PostalCode w:st="on">50011-1020</st1:PostalCode>
 <st1:country-region w:st="on">USA</st1:country-region></st1:place> <o:p></o:p></p>

<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'>Phone: (515)-294-3872 <o:p></o:p></span></font></p>

<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'>Fax:&nbsp;&nbsp;&nbsp;&nbsp; (515)-294-1337 <o:p></o:p></span></font></p>

<p class=MsoNormal style='mso-margin-top-alt:0in;margin-right:0in;margin-bottom:
12.0pt;margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'>Email: ihilal@iastate.edu<o:p></o:p></span></font></p>

<p class=MsoNormal style='margin-left:.5in'><font size=3 face="Times New Roman"><span
style='font-size:12.0pt'><a href="http://www.public.iastate.edu/~ihilal/"
eudora=autourl>http://www.public.iastate.edu/~ihilal/</a> <o:p></o:p></span></font></p>

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