<html>
<head>
<meta http-equiv=Content-Type content="text/html; charset=us-ascii">
<meta name=Generator content="Microsoft Word 10 (filtered)">
<style>
<!--
/* Font Definitions */
@font-face
{font-family:Wingdings;
panose-1:5 0 0 0 0 0 0 0 0 0;}
@font-face
{font-family:"MS Mincho";
panose-1:2 2 6 9 4 2 5 8 3 4;}
@font-face
{font-family:"\@MS Mincho";
panose-1:2 2 6 9 4 2 5 8 3 4;}
/* Style Definitions */
p.MsoNormal, li.MsoNormal, div.MsoNormal
{margin:0in;
margin-bottom:.0001pt;
font-size:10.0pt;
font-family:"Times New Roman";}
a:link, span.MsoHyperlink
{color:blue;
text-decoration:underline;}
a:visited, span.MsoHyperlinkFollowed
{color:purple;
text-decoration:underline;}
span.EmailStyle17
{font-family:Arial;
color:windowtext;}
@page Section1
{size:8.5in 11.0in;
margin:1.0in 1.0in 1.5in 1.5in;}
div.Section1
{page:Section1;}
/* List Definitions */
ol
{margin-bottom:0in;}
ul
{margin-bottom:0in;}
-->
</style>
</head>
<body lang=EN-US link=blue vlink=purple>
<div class=Section1>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Hello Everyone-</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>I am desperately, and rather unsuccessfully, trying to find
a better way to look at eGFP in frozen kidney sections from mice.</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>I have read a wealth of info on histonet regarding use of
GFP antibodies, and I have tried a number of methods, but I am still not having
any luck.</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>I have tried the following:</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in'><font size=2
face=Arial><span style='font-size:10.0pt;font-family:Arial'>(1)<font size=1
face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font face=Arial><span style='font-family:Arial'>looking
directly at eGFP in frozen sections (sections are just a few weeks old) right
after 15 min fixation with 4% paraformaldehyde (fresh 16% paraformaldehyde
diluted in PBS), followed by washes in PBS</span></font><font face=Wingdings><span
style='font-family:Wingdings'>à</span></font><font face=Arial><span
style='font-family:Arial'> no GFP is observed</span></font></p>
<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in'><font size=2
face=Arial><span style='font-size:10.0pt;font-family:Arial'>(2)<font size=1
face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font face=Arial><span style='font-family:Arial'>looking
directly at eGFP in same frozen sections without fixation, coverslipping in PBS
</span></font><font face=Wingdings><span style='font-family:Wingdings'>à</span></font><font
face=Arial><span style='font-family:Arial'> no GFP is observed</span></font></p>
<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in'><font size=2
face=Arial><span style='font-size:10.0pt;font-family:Arial'>(3)<font size=1
face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font face=Arial><span style='font-family:Arial'>fixation
in paraformaldehyde as described above followed by a GFP antibody conjugated to
a fluorochrome </span></font><font face=Wingdings><span style='font-family:
Wingdings'>à</span></font><font face=Arial><span style='font-family:
Arial'> very little signal is observed</span></font></p>
<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in'><font size=2
face=Arial><span style='font-size:10.0pt;font-family:Arial'>(4)<font size=1
face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font face=Arial><span style='font-family:Arial'>same
fixation, followed by incubation with GFP antibody, follwed by biotinylated
anti-rabbit, and finally by streptavidin-Alexa 568</span></font></p>
<p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in'><font size=2
face=Symbol><span style='font-size:10.0pt;font-family:Symbol'>·<font
size=1 face="Times New Roman"><span style='font:7.0pt "Times New Roman"'>
</span></font></span></font><font face=Arial><span style='font-family:Arial'>This
method resulted in EXTREMELY high background. I tried to block endogenous
biotin by using both the egg/milk protocol and biotin/avidin drops from Vector
Labs, both with little success.</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>I mount my slides with Flouromount, do NOT seal with nail
polish, and I do not do antigen retrieval (this is not necessary for frozen
sections, right?).</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Any input would be greatly appreciated!</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Thank you!</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Kristen Rothammer</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>St. Jude Children’s </span></font><font face=Arial><span
style='font-family:Arial'>Research</span></font><font face=Arial><span
style='font-family:Arial'> </span></font><font face=Arial><span
style='font-family:Arial'>Hospital</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'>Department of Biochemistry</span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'><a href="mailto:Kristen.rothammer@stjude.org">Kristen.rothammer@stjude.org</a></span></font></p>
<p class=MsoNormal><font size=2 face=Arial><span style='font-size:10.0pt;
font-family:Arial'> </span></font></p>
</div>
</body>
</html>