basic question about immunohistochemistry

From: Steve Mullen (mullensf@vet.vet.purdue.edu)
Date: Wed Aug 30 2000 - 19:15:46 EST


Dear cytometrists,

I am trying to work up an immunohistochemical protocol trying to determine
the presence of an enzyme with a double-labelled antibody system. The
secondary Ab is labelled with Cy-3. My signals are very weak, even in the
positive control.

My basic question is this. Am I just dreaming that I will get a strong
enough signal on individual cells without a signal amplification system?
The protocol I am following suggests it can be done on cells adhered to
slides, so I assume it is possible.

Furthermore, I am kind of new to this and the protocol calls for rinsing
after the secondary Ab incubation step. What should I be looking out for
when it comes to avoiding washing out the antibodies. I have been
consulting texts and they are some help but I thought discussing this with
a live person would be more fruitful.

I appreciate the expertise and hope I don't get chastised for asking a
question that "could be" answered by a literature search.


Thanks in advance.

Steve

Steven F. Mullen
Graduate Fellow/Research Assistant
Purdue University
Department of Veterinary Clinical Sciences
Cryobiology Laboratory
Lynn Hall, G242
West Lafayette, Indiana  47907-1398
Phone: 765-494-0336
Fax: 765-496-1108
mullensf@vet.purdue.edu



This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:30 EST