[Histonet] Stripping antibodies

[John Kiernan]

With quantum dots and a modern fluorescence microscope with "channels" it should be possible to label 2 or 3 antigens simultaneously, if you have all the right primaries and labelled secondaries. The company that sells you the quantum dot labelled secondaries or other amplification reagents should be in a position to tell you exactly what to do. Do they refund your hard-earned grant money if their expensive product fails to perform as advertized? 
 
Immunohistochemistry with non-fluorescent colours (available in brown, black, red and various blues) has advantages: 
(a) You do not need a fluorescence microscope. Modern fluorescence microscopes are very expensive; the old ones from the 1960s are no longer be good enough to provide publication-quality pictures.
(b) Immunostained slides can be kept for many years in boxes at room temperature.
(c) The colours do not fade in permanently mounted preparations. 
(c) You are not bound to use a commercially sold kit, which may not be optimized for your research.
 
If you go by the book (= any book with references that you can follow up), you will do your multiple immunostains intelligently. Never follow a set of instructions without knowing or questioning the reason for ever step. 
 
John Kiernan 
Anatomy, UWO 
London, Canada. 
= = = 
 On 30/10/11, Claire Weston <cweston <@t> valasciences.com> wrote:

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