Re: IHC titer

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From:Tim Morken <> (by way of histonet)
To:histonet <>
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The best place to start is with any product info you get with the
anitbody. Use the recommended dilution as a starting point and adjust it
from there.

If you don't have any info (as it usually is in our case) we do titers
over a broad range to start with and then narrow the range as we gain
information about the antibody.

For instance, we might do 1:50, 1:500 and 1:1000 to start. Depending on
the results, we would do the next titer series near one of those
dilutions. For instance, if it works better at 1:500, our next series
might be 1:300, 1:500, 1:700. From those results we sould narrow it

Alternativly, you could do a doubling series in which you start at 1:20
and then double the dilution up to 1:1000 or more all in one run.

We generally start with digestion with Protienase K and only try antigen
retrieval if no positive results are obtained.

We keep records of the trials and keep the experiment that shows the
best dilution as a reference. That experiment is noted in the antibody

For hardware it would be best to have several pipets. They will cost you
in the neighborhood of USD 150 to 250 each but they are well worth the
cost since they allow you to make very small volumnes of expensive

We have several in the following ranges:
0.5 to 10 ul
10 to 100 ul
100 to 1000 ul
200 to 100 ul
50 to 200 ul
10 to 50 ul

These are checked for calibration annually.

Tim Morken, B.A., EMT(MSA), HTL(ASCP)
Infectious Disease Pathology
Centers for Disease Control
1600 Clifton Rd.
Atlanta, GA 30333


FAX:  (404)639-3043

----Original Message Follows----
Date: Wed, 24 Feb 1999 21:51:54 -0500 (EST)
Subject: IHC titer

Hi Histoinans !
   I would like some help with titering my immunohistochemical primary
antibodies, please.
   How many dilutions and in what ranges. What does an economical
pipette =
this procedure cost? Do you use titer records & retain titer slides? Do
adjust antibodies on special request from the Pathologist to demonstrate
specifically =93hot=94 markers?
   I=92ve told my Pathologists that we need to invest in the pipette for
titering procedure. I keep records of the dilutions we arrive at and
make =
Pathologist sign off on each lot of primary antibody.
   Thank You for any help

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