Just a thought.  Could it be that the second round of antigen retrieval is
washing away the first substrate?  Have you checked that the chromagen from
the first antibody is still there after the second round of HIER, and before
the second antibody.  

Did you try different orders of the detection?  I would think that the
nuclear antigen detection should be first, or you may be losing it.

This is an interesting problem to me, and I will be very interested in the
solution(s).  It is the research scientist in me.

Noelle Patterson, M.S.
NN-TAB
Bethesda, Md.  20889

stressed is desserts spelled backwards.  



> -----Original Message-----
> From:	C & J Haley [SMTP:haley@primary.net]
> Sent:	Wednesday, March 01, 2000 9:00 PM
> To:	HistoNet Server
> Subject:	Double IHC question with clarification
> 
> Hello again: 
> 
> Okay, so I didn't give enough information to get enough information. 
> 
> Linda, Patsy, Danielle:      I know, usually if they require the same
> pretreatment, there's no need  for repeating it.  These guys need to have
> their own round of EDTA HIER :( . Believe me, I've tried cutting one out,
> altering one's time, etc.  I have optimized them separately.  Separately,
> they look great.   But I need to have the double staining on the same
> slide.    And that leads me to the next question. 
> 
> Cynthia:  So why would anybody need to put two antibodies that work best
> alone on the same slide?  The answer is simply:  pesky Pathologist, pretty
> pictures for a publication, and a nice little challenge.  I should  also
> mention that one antibody is a nuclear marker and the other is a nice
> little membranous marker.  Co-expression on the same cell  should make for
> some nice photos. And to answer your  questions:  yes(substate),
> yes(times/temps), and no because that shows that you know the many problem
> parameters of IHC. 
> 
> Diagnostically, most double  immunostains are not practical.  For
> researchers it can serve many purposes (co-expression, working with
> material limitations, antibody specificity, etc.).  Also, most  Histotechs
> (especially those on the Histonet)  like a challenge and a beautifully
> stained slide. So, I'll continue to "tweak" my procedure and wait for more
> responses or questions. 
>   
> 
> Thanks in advance, 
> 
> Jane Haley 
> Barnes-Jewish Hospital 
> St. Louis, Mo. 
>   
>   
>   
>