[Histonet] Stripping antibodies

Claire Weston cweston <@t> valasciences.com
Sun Oct 30 13:58:19 CDT 2011


<DIV style="font-family:Arial, sans-serif; font-size:10pt;"><DIV>Thank you everyone for your advice, I really appreciate it!&nbsp; I am doing a multiplex immunofluorescent assay (4 channels) and I am planning to strip the antibodies and reprobe with a second round of antibodies.&nbsp; The antibodies are labelled with quantum dots so I am not sure how that will influence things, but luckily I won't have the DAB problem several of you mentioned.&nbsp; I think I will try several of these techniques and evaluate which works the best.</DIV>
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<DIV>Thanks again for all your help,</DIV>
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<DIV>Claire&nbsp; <BR><BR>--- jkiernan <@t> uwo.ca wrote:<BR><BR>From: John Kiernan &lt;jkiernan <@t> uwo.ca&gt;<BR>To: histonet <@t> lists.utsouthwestern.edu, cweston <@t> valasciences.com<BR>Cc: Amos Brooks &lt;amosbrooks <@t> gmail.com&gt;<BR>Subject: Re: [Histonet] Stripping antibodies<BR>Date: Sun, 30 Oct 2011 12:09:58 -0400<BR><BR></DIV>
<DIV><SPAN>&nbsp; 
<DIV>Antibodies&nbsp;are quite easily removed by acidity - pH 1 to 2.&nbsp;This also removes enzyme-antibody conjugates.&nbsp;The coloured products of the enzyme histochemical reactions are generally not affected. The brown oxidation product of DAB (from peroxidase) and the blue products from bromochloroindoxyl/tetrazolium methods (for alkaline phosphatase) are very stable.&nbsp;This is good: you can remove all the primary and enzyme-labelled antibodies without losing track of the stained antigen in the section.&nbsp;A second immunostaining, with a differently coloured end-product can follow.&nbsp; If you use AEC as a&nbsp;peroxidase chromogen (red), it should be for the&nbsp;last immunohistochemical procedure, and you will have to use an aqueous mounting medium. The AEC oxidation product dissolves in alcohol and other organic solvents.</DIV>
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<DIV>Amos suggests a strong oxidation (acid permanganate followed by oxalic acid to remove brown manganese dioxide) to get rid of the insoluble brown oxidation product of DAB. Is this what you want?&nbsp; This&nbsp;oxidation converts the sulphur-containing amino acids, including cystine&nbsp;-S-S- links, to sulphonic acids, which attract cationic dyes even at pH 1.&nbsp;This oxidation couuld seriously modify the epitopes for the next applied primary antibody.</DIV>
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<DIV>Multicolour immunohistochemistry has been a developed technology for several years. Any lab needing to do such work&nbsp;needs to take training courses (hundreds of dollars) or buy a book (tens of dollars). &nbsp;The one by Chris van der Loos, Immunoenzyme Multiple Staining Methods, Oxford, UK: Bios, &nbsp;1999, is very good.</DIV>
<DIV>&nbsp;</DIV>
<DIV>John Kiernan</DIV>
<DIV>Anatomy, UWO&nbsp;</DIV>
<DIV>London, Canada&nbsp;</DIV>
<DIV>= = = =</DIV>On 29/10/11, <B>Amos Brooks </B>&lt;amosbrooks <@t> gmail.com&gt; wrote:</SPAN> 
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<DIV>Hi,<BR>&nbsp;&nbsp;&nbsp; Stripping sites are usually found in the more seedy areas of large<BR>cities ... Oh wait you meant ... nevermind :-) If you developed the reaction<BR>with DAB, you may be in a difficult position. DAB is a really strong<BR>reaction. You might try a Mallory bleach to remove it. 0.3% sulfuric acid in<BR>5% potassium premanganate for 5 min followed by 5% oxalic acid until it is<BR>clear (usually a couple of minutes). Unfortunately, I am not sure what<BR>effect this will have on the epitopes you are looking for, but this is<BR>usually what is used to clean precipitated DAB from instruments it should<BR>work on the tissue too.<BR>&nbsp;&nbsp;&nbsp; If you use another chromogen such as AEC it is much easier (to many<BR>people's dismay) to remove this end product. Using alkaline phosphatase will<BR>be easy to remove the chromogens as well. It would also be very easy to do<BR>this with fluorescent tags too. Just remove the coverslip and dip the slide<BR>in ethanol and it will remove the colored end product. Then just start over<BR>for the new antigen.<BR><BR>Good luck,<BR>Amos<BR><BR>On Fri, Oct 28, 2011 at 11:50 AM, &lt;histonet-request <@t> lists.utsouthwestern.edu<BR>&gt; wrote:<BR><BR>&gt; Message: 2<BR>&gt; Date: Thu, 27 Oct 2011 10:30:40 -0700<BR>&gt; From: "Claire Weston" &lt;cweston <@t> valasciences.com&gt;<BR>&gt; Subject: [Histonet] Stripping antibodies<BR>&gt; To: &lt;histonet <@t> lists.utsouthwestern.edu&gt;<BR>&gt; Message-ID: &lt;002401cc94ce$2619ca60$724d5f20$@com&gt;<BR>&gt; Content-Type: text/plain;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; charset="us-ascii"<BR>&gt;<BR>&gt; Hi!<BR>&gt;<BR>&gt;<BR>&gt;<BR>&gt; Does anyone have experience in IHC stripping antibodies from a tissue<BR>&gt; section and re-probing with different antibodies?&nbsp; What is the best way to<BR>&gt; do that?&nbsp; If you could share your stripping protocol or experience I would<BR>&gt; appreciate it - thanks!<BR>&gt;<BR>&gt;<BR>&gt;<BR>&gt; Claire<BR>&gt;<BR>_______________________________________________<BR>Histonet mailing list<BR>Histonet <@t> lists.utsouthwestern.edu<BR><A href="http://lists.utsouthwestern.edu/mailman/listinfo/histonet">http://lists.utsouthwestern.edu/mailman/listinfo/histonet</A><BR></DIV></BLOCKQUOTE>
<DIV>&nbsp;</DIV>&nbsp;</DIV>
<DIV>&nbsp;</DIV><SPAN>On 29/10/11, <B>Amos Brooks </B>&lt;amosbrooks <@t> gmail.com&gt; wrote:</SPAN> 
<BLOCKQUOTE style="BORDER-LEFT: #00f 1px solid; PADDING-LEFT: 13px; MARGIN-LEFT: 0px">
<DIV>Hi,<BR>&nbsp;&nbsp;&nbsp; Stripping sites are usually found in the more seedy areas of large<BR>cities ... Oh wait you meant ... nevermind :-) If you developed the reaction<BR>with DAB, you may be in a difficult position. DAB is a really strong<BR>reaction. You might try a Mallory bleach to remove it. 0.3% sulfuric acid in<BR>5% potassium premanganate for 5 min followed by 5% oxalic acid until it is<BR>clear (usually a couple of minutes). Unfortunately, I am not sure what<BR>effect this will have on the epitopes you are looking for, but this is<BR>usually what is used to clean precipitated DAB from instruments it should<BR>work on the tissue too.<BR>&nbsp;&nbsp;&nbsp; If you use another chromogen such as AEC it is much easier (to many<BR>people's dismay) to remove this end product. Using alkaline phosphatase will<BR>be easy to remove the chromogens as well. It would also be very easy to do<BR>this with fluorescent tags too. Just remove the coverslip and dip the slide<BR>in ethanol and it will remove the colored end product. Then just start over<BR>for the new antigen.<BR><BR>Good luck,<BR>Amos<BR><BR>On Fri, Oct 28, 2011 at 11:50 AM, &lt;histonet-request <@t> lists.utsouthwestern.edu<BR>&gt; wrote:<BR><BR>&gt; Message: 2<BR>&gt; Date: Thu, 27 Oct 2011 10:30:40 -0700<BR>&gt; From: "Claire Weston" &lt;cweston <@t> valasciences.com&gt;<BR>&gt; Subject: [Histonet] Stripping antibodies<BR>&gt; To: &lt;histonet <@t> lists.utsouthwestern.edu&gt;<BR>&gt; Message-ID: &lt;002401cc94ce$2619ca60$724d5f20$@com&gt;<BR>&gt; Content-Type: text/plain;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; charset="us-ascii"<BR>&gt;<BR>&gt; Hi!<BR>&gt;<BR>&gt;<BR>&gt;<BR>&gt; Does anyone have experience in IHC stripping antibodies from a tissue<BR>&gt; section and re-probing with different antibodies?&nbsp; What is the best way to<BR>&gt; do that?&nbsp; If you could share your stripping protocol or experience I would<BR>&gt; appreciate it - thanks!<BR>&gt;<BR>&gt;<BR>&gt;<BR>&gt; Claire<BR>&gt;<BR>_______________________________________________<BR>Histonet mailing list<BR>Histonet <@t> lists.utsouthwestern.edu<BR><A href="http://lists.utsouthwestern.edu/mailman/listinfo/histonet">http://lists.utsouthwestern.edu/mailman/listinfo/histonet</A><BR></DIV></BLOCKQUOTE>
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