<html><base href="https://freemail.web.de/"/><style>p {margin: 0px}</style><body style='font-size:9pt; font-family:Verdana; font-family: Verdana' ><P>Dear All,</P><P> I have a problem with IHC staining(using Perioxdase,AEC substrate)) on 30 um thick mouse lymph node frozen sections. After staining part of section is washed away and the morphology of the section is poor. Can someone help me?</P><P> My protocol is following:</P><P> </P><P class=MsoNormal><SPAN style="mso-spacerun: yes"> </SPAN>IHC<SPAN style="mso-spacerun: yes"> </SPAN>staining protocol</P><P class=MsoNormal><?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" /><o:p> </o:p></P><OL style="MARGIN-TOP: 0cm" type=1><LI class=MsoNormal style="mso-list: l1 level1 lfo1; tab-stops: list 36.0pt">Cut 30 um frozen sections.</LI><LI class=MsoNormal style="mso-list: l1 level1 lfo1; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Fix in cold methanol <SPAN style="mso-spacerun: yes"> </SPAN>for <SPAN style="COLOR: red">3</SPAN> minutes, then in cold aceton for <SPAN style="COLOR: red">3 </SPAN>minutes.<o:p></o:p></SPAN></LI></OL><P class=MsoNormal style="MARGIN-LEFT: 36pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Air dry for <SPAN style="COLOR: red">1 </SPAN>hour.<o:p></o:p></SPAN></P><OL style="MARGIN-TOP: 0cm" type=1 start=3><LI class=MsoNormal style="mso-list: l1 level1 lfo1; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Put the section in PBS for <SPAN style="COLOR: red">10</SPAN> minutes.<o:p></o:p></SPAN></LI><LI class=MsoNormal style="mso-list: l1 level1 lfo1; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Block with 1%FCS/PBS <SPAN style="mso-spacerun: yes"> </SPAN><SPAN style="COLOR: red">100 ul</SPAN> for <SPAN style="COLOR: red">20</SPAN> minutes at room<SPAN style="mso-spacerun: yes"> </SPAN>temperature.<o:p></o:p></SPAN></LI><LI class=MsoNormal style="mso-list: l1 level1 lfo1; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Endogenous biotin blocking:<o:p></o:p></SPAN></LI></OL><P class=MsoNormal style="MARGIN-LEFT: 72pt; TEXT-INDENT: -18pt; mso-list: l1 level2 lfo1; tab-stops: list 72.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-list: Ignore">(1)<SPAN style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN></SPAN><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Incubate slice in egg solution for <SPAN style="COLOR: red">15 </SPAN>minutes.<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 72pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Egg solution: <SPAN style="COLOR: red">25 </SPAN>ml egg white ,<SPAN style="COLOR: red">75</SPAN> ml distilled water.<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 72pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Wash with 2 changes of<SPAN style="mso-spacerun: yes"> </SPAN>distilled water.<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 72pt; TEXT-INDENT: -18pt; mso-list: l1 level2 lfo1; tab-stops: list 72.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-list: Ignore">(2)<SPAN style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN></SPAN><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Incubate slice in the milk(fat free) for <SPAN style="COLOR: red">15</SPAN> minutes at room temperature.<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 54pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-spacerun: yes"> </SPAN>Wash with 2 changes of distilled water.<o:p></o:p></SPAN></P><OL style="MARGIN-TOP: 0cm" type=1 start=6><LI class=MsoNormal style="mso-list: l1 level1 lfo1; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Add diluted first antibody.<o:p></o:p></SPAN></LI></OL><P class=MsoNormal style="MARGIN-LEFT: 36pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">For example :CD3-biotin<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 18pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><o:p> </o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Incubate <SPAN style="COLOR: red">2 </SPAN>hours at room temperature or at <SPAN style="COLOR: red">4 °C</SPAN><SPAN style="mso-spacerun: yes"> </SPAN>overnight.<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Wash in PBS for <SPAN style="COLOR: red">30 </SPAN>seconds, <SPAN style="COLOR: red">3</SPAN> <SPAN style="mso-spacerun: yes"> </SPAN>minutes and <SPAN style="COLOR: red">10</SPAN> minutes three times.<o:p></o:p></SPAN></P><OL style="MARGIN-TOP: 0cm" type=1 start=7><LI class=MsoNormal style="mso-list: l1 level1 lfo1; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">endogenous peroxidase blocking:<o:p></o:p></SPAN></LI></OL><P class=MsoNormal style="MARGIN-LEFT: 36pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">The solution : <SPAN style="COLOR: red">0,3%</SPAN><SPAN style="mso-spacerun: yes"> </SPAN>H</SPAN><SPAN lang=EN-GB style="FONT-SIZE: 8pt; mso-ansi-language: EN-GB">2</SPAN><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">O</SPAN><SPAN lang=EN-GB style="FONT-SIZE: 8pt; mso-ansi-language: EN-GB">2</SPAN><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-spacerun: yes"> </SPAN>,<SPAN style="COLOR: red">0,25</SPAN>% NaN3<SPAN style="mso-spacerun: yes"> </SPAN>in PBS<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-spacerun: yes"> </SPAN>Incubate the section in this solution for <SPAN style="COLOR: red">half </SPAN><SPAN style="mso-spacerun: yes"> </SPAN>hour.<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-spacerun: yes"> </SPAN>Rinse with water for <SPAN style="COLOR: red">2</SPAN> minutes.<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-spacerun: yes"> </SPAN>Wash in PBS<SPAN style="mso-spacerun: yes"> </SPAN>for<SPAN style="mso-spacerun: yes"> </SPAN><SPAN style="COLOR: red">5</SPAN> minutes.<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo2; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-list: Ignore">8<SPAN style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN></SPAN><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Block with 1%FCS/PBS for another <SPAN style="COLOR: red">20</SPAN> minutes at room temperature.<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 15pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">9. Add<SPAN style="mso-spacerun: yes"> </SPAN>second antibody<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 15pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-spacerun: yes"> </SPAN>for example streptavidin-PO<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 15pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-spacerun: yes"> </SPAN><o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 15pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-spacerun: yes"> </SPAN>Incubate for <SPAN style="COLOR: red">4</SPAN> hours at 4 °C..<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 15pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-spacerun: yes"> </SPAN><SPAN style="mso-spacerun: yes"> </SPAN>Wash with PBS for <SPAN style="COLOR: red">1 ,3 ,10</SPAN> minutes for three times.<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo2; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-list: Ignore">9<SPAN style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN></SPAN><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Add substrate <SPAN style="COLOR: red">300 </SPAN>ul for each section, incubate at <SPAN style="COLOR: red">37°C</SPAN> surface for <SPAN style="COLOR: red">half <SPAN style="mso-spacerun: yes"> </SPAN></SPAN>hour.<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 33pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Substrate solution:<SPAN style="mso-spacerun: yes"> </SPAN><SPAN style="COLOR: red">25</SPAN> ul AEC stock solution<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 33pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-spacerun: yes"> </SPAN><SPAN style="COLOR: red">1</SPAN> ml Na-Acetate buffer(5,4)<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 33pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-spacerun: yes"> </SPAN><SPAN style="COLOR: red">1</SPAN>ul H</SPAN><SPAN lang=EN-GB style="FONT-SIZE: 8pt; mso-ansi-language: EN-GB">2</SPAN><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">O</SPAN><SPAN lang=EN-GB style="FONT-SIZE: 8pt; mso-ansi-language: EN-GB">2</SPAN><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 33pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Attention: Make this solution just before use,protect it from light..<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 33pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Wash in PBS for <SPAN style="COLOR: red">30</SPAN> seconds,<SPAN style="COLOR: red">3 </SPAN>minutes<SPAN style="mso-spacerun: yes"> </SPAN>and <SPAN style="COLOR: red">10</SPAN> minutes for three times.<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo2; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-list: Ignore">10<SPAN style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN></SPAN><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Dry on 37°C surface for <SPAN style="COLOR: red">5 </SPAN>minutes.<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo2; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-list: Ignore">11<SPAN style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN></SPAN><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-spacerun: yes"> </SPAN>Mount with<SPAN style="mso-spacerun: yes"> </SPAN><SPAN style="COLOR: red">175 </SPAN>ul Kaser’s galatine using 24x60 cover<SPAN style="mso-spacerun: yes"> </SPAN>glass.<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 15pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><o:p> </o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo2; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-list: Ignore">12<SPAN style="FONT: 7pt 'Times New Roman'"> </SPAN></SPAN></SPAN><SPAN lang=EN-GB style="mso-ansi-language: EN-GB">Read the slides.<o:p></o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo2; tab-stops: list 36.0pt"> </P><P class=MsoNormal style="MARGIN-LEFT: 36pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo2; tab-stops: list 36.0pt"> Will adding Triton <SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><o:p> or tween 20 help ?</o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo2; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><o:p> Thank you.</o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo2; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><o:p>Bin ma</o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo2; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><o:p>German National research Centre ofBiotechnology</o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo2; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><o:p>Braunschweig</o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo2; tab-stops: list 36.0pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><o:p>Germany</o:p></SPAN></P><P class=MsoNormal style="MARGIN-LEFT: 36pt"><SPAN lang=EN-GB style="mso-ansi-language: EN-GB"><SPAN style="mso-spacerun: yes"> </SPAN><o:p></o:p></SPAN></P></body></html>
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