[Histonet] using predilutes

[Orr, Rebecca]

When I have encountered a prediluted antibody that works best when I
dilute it out further, I go ahead and just find the concentrate and
dilute out myself.

I hesitate adding more diluent to a product that already has been
diluted. Granted even our concentrates are not "full strength" even
concentrates are diluted by the time they get to us. But for me in a
clinical lab, I believe that if a predilute must be diluted out further,
why bother with it?  I am adding another variable to the mixture.  I
don't know what diluent the manufacturer is using...what pH, whether
it's pbs or not... what preservatives... ok ok, yes I can look at the
data sheet and test the pH of the predilute to figure all that
out...like I said, why bother? I'd have to have another type of
validation to justify diluting out this type of product. I'd have to
somehow document why and how I am not following manufacturer's
recommendations on a prediluted product... or am I just in over kill
mode here?

If a predilute is over staining,  I decide if I want to back off
incubation time..rethink my detection or check over the HIER.

I ran into this with  the prediluted SMA from Biocare...it was
overstaining everything!  I suspect it's the Mach 4 that is just crazy
hot...

Plus I was running HIER... once I actually READ the data sheet, I saw
that they don't recommend HIER!

So, with that said, I ended up buying the concentrate and running it
about 1:500 NO HIER.  All better now.

 

I guess the point I'm trying to make is to keep trying when you get over
stained results or no results...sometimes it's  just a matter of
combining the right detection/antibody.  Maybe since I've been in tech
support I understand how valuable the manufacturer's tech support
staffers really are in helping out in these situations.

Ok I'll stop now...I've had way too much coffee this morning.

 

Becky Orr

Evanston Hospital

 

Message: 14

Date: Fri, 06 Apr 2007 16:41:20 -0400

From: "Richard Cartun" <Rcartun <@t> harthosp.org>

Subject: Re: [Histonet] ready to use antibody

To: <histonet <@t> lists.utsouthwestern.edu>,  "Kristopher Kalleberg"

      <Kristopher.Kalleberg <@t> unilever.com>

Message-ID: <4616783002000077000052A0 <@t> gwmail.harthosp.org>

Content-Type: text/plain; charset=US-ASCII

 

We don't use many "ready-to-use" (RTU) antibodies, but we have found
that we can usually dilute the ones that we do use 1:5, 1:10, 1:20,
1:50, or maybe even 1:100 for optimal staining.  Obviously, the dilution
will be determined by the sensitivity of your detection system and the
length of your primary antibody incubation.  Have you tried different
tissue pretreatments?

 

Richard

 

Richard W. Cartun, Ph.D.

Director, Immunopathology & Histology

Assistant Director, Anatomic Pathology

Hartford Hospital

80 Seymour Street

Hartford, CT  06102

(860) 545-1596

(860) 545-0174 Fax

 

>>> "Kalleberg, Kristopher" <Kristopher.Kalleberg <@t> unilever.com> 04/06/07


>>> 2:21 PM >>>

Has anyone ever used the ready to use antibodies which are prediluted?
They are offerred by Lab Vision/Neomarkers.  Is there any special
protocol for these antibodies?  I have run an experiment twice with this
prediluted antibody and have had no results.  Technical support was
unable to help me with these items.Any help or information will be
greatly appreciated.  Thank you.