[Histonet] RE: Elution reagent for Double Staining
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.
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
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
Date Tue, 23 Nov 2004 08:38:59 -0700
To Amy Porter ,
Subject Re: [Histonet] Elution reagent for Double Staining
Talk to Chris van der Loos - he has done this and actually feels this
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
. There are problems with elution. One is
antibody is bound so strongly to antigen, it will not elute. Another
is damage to antigens with elution reagents. Chris is a master at
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
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
>double immunostaining using peroxidase and phosphatase systems
>together? I have looked and gotten a variety of answers, I would like
>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
Histonet mailing list
<< Previous Message | Next Message >>