Re: [Histonet] IHC - weak signals

From:"Robyn Vazquez"

I use the Ted Pella microwave for tissue processing.  Does anyone have a good procedure for this.  It is a hit and miss sometimes.  I don't use JFC anymore, but instead I use reagent alcohol.  The instructions I was given was if I process the same day, I let the tissue sit in non-buffered formalin for ˝ hour then process 15-20 min (which had to be cut down to 2-3 min), then rinse 95% for 2 min, then into 100% absol 15-20min(down to 2-3 min) 65 C, JFC sol the same absolute but at 70 c.  I changed the JFC sol for reagent alc still no change.  The tissue does work out most of the time, but it's those times that it doesn't, makes it frustrating.  Then into paraffin at 83 C the same time down to 2-3 min.  My tissue would shrink and be crunchie.  I tried the saline fixative yesterday with extra tissue.  It shrank 50%, but was not crunchie.  Do I have my paraffin to hot? thanks for your input....
Robyn Vazquez

>>> "Greg Dobbin"  9/24/2004 12:02:05 PM >>>

Hi Jason,
Post more details about your procedure. The chromagen step is
only one of many steps that can contribute to signal strength, and at
that, not the most likely place to start. Tell the group what you are
staining for, with frozens or paraffin sections, how long and at what
temp the primary is applied, with what detection system, if any
retreival methods are being employed, etc. We need the whole
story before we can offer any help.

From:               "jason m" 
BCC to:             Date sent:          Fri, 24 Sep 2004 07:19:25 -0700
Copies to:          Subject:            [Histonet] IHC - weak signals

> Does anyone have advice for amplifying weak signals from IHC? Is there a
> detection system better than DAB which can make the signal more noticable?
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Greg Dobbin
Pathology Lab
Atlantic Veterinary College, U.P.E.I.
550 University Ave.
Charlottetown, P.E.I.
Canada,  C1A 4P3
Phone: (902)566-0744
Fax: (902)566-0851
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