[Histonet] RE: Elution reagent for Double Staining

[C.M. van der Loos]

Dear Amy,
Gayle is right: I don't regard sequential double staining with elution 
in between as a very safe way of double staining. Only in case you wish 
to stain to different cell populations, and you are sure there is no co-
localization involved, you may try sequential double staining. The 
DakoCyto EnVision Doublestain kit is worthwhile to test. 
The so called "doublestain block" is equal to the low pH glycin-HCl 
buffer and isn't working either.  
Some remarks: 
1. Sequential double staining only works because of unique effective 
sheltering of DAB chromogen. As far as I know there are no other 
enzymatic chromogens with this sheltering capacity. Always apply DAB 
after the 1st staining sequence!!!!! 
2. You may try to remove reagents of the 1st staining sequence using 
glycing-HCl buffer (0.1 M, pH2.2) however high affinity primaries will 
stay at your tissue section anyway. In the DakoCyto EnVision 
Doublestain kit the "doublestain block" is equal to the low pH glycin-
HCl buffer and isn't working either for removing high affinity 
primaries. Always test your double staining with/without the elution 
step. 
3. Glycin-HCl buffer may damage the tissue morphology of an acetone-
fixed cryostat section. Don't do this.
4. Again: sequential double staining technique is not for visualizing 
co-localization by mixed-colors! Furthermore, the brown-red (or brown-
blue if you wish) color combi is very poor for visualizing a 
distinguishable mixed-color. Don't do this.
5. If you are immunostaining two antigens that are at very closely 
together you may end up in trouble: DAB after the 1st staining sequence 
will effectively shelter the 2nd antigen too.
Hope these do-s and don't-s work for you.

Chris van der Loos, PhD
Dept. of Pathology
Academical Medical Center
Amsterdam - The Netherlands 

----- Original Message ----- 
>From  Gayle Callis <gcallis <@t> montana.edu> 
Date  Tue, 23 Nov 2004 08:38:59 -0700 
To  Amy Porter <portera203 <@t> yahoo.com>, 
Histonet <@t> lists.utsouthwestern.edu 
Subject  Re: [Histonet] Elution reagent for Double Staining 

Talk to Chris van der Loos - he has done this and actually feels this 
does 
NOT work very well.  There are other ways to get excellent results with 
double IHC and not use elution.  He can be reached at "C.M. vander 
Loos" 
<c.m.vanderloos <@t> amc.uva.nl>.  There are problems with elution.  One is 
the 
antibody is bound so strongly to antigen, it will not elute.  Another 
is  damage to antigens with elution reagents.  Chris is a master at 
double 
staining and taught this in Toronto this past year - look for him to do 
this again in Fort Lauderdale, NSH symposium 2005 - he did discuss 
elution 
and how to get around it.
A good visit with him may help you with your double staining.

At 07:33 AM 11/23/2004, you wrote:
>Anyone out there willing to share what they use for elution reagent 
during 
>double immunostaining using peroxidase and phosphatase systems 
>together?  I have looked and gotten a variety of answers, I would like 
to 
>know what actually works for people.  Thanks in advance -
>
>Amy S.Porter, HT(ASCP)
>Michigan State University
>Department of Physiology
>Division of Human Pathology
>College of Human Medicine
>portera203 <@t> yahoo.com