Re: IHC titer

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From:vandeplas@aurion.nl (Peter van de Plas) (by way of histonet)
To:histonet <histonet@magicnet.net>
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Verrucous wrote:
>   I would like some help with titering my immunohistochemical primary
>antibodies, please.
>   How many dilutions and in what ranges. What does an economical pipette for
>this procedure cost? Do you use titer records & retain titer slides? Do you
>adjust antibodies on special request from the Pathologist to demonstrate
>specifically hot markers?
>   Ive told my Pathologists that we need to invest in the pipette for proper
>titering procedure. I keep records of the dilutions we arrive at and make the
>Pathologist sign off on each lot of primary antibody.
---------------------------------
The dilution of a primary antibody that will give you the optimal signal to
noise ratio depends on a lot of factors, e.g. concentration of your stock,
binding affinity of the primary antibody and the detection system you are
using. The following (my approach) might be of help:

In general you incubate with a primary antibody concentration between 10
and 1 g/ml. When you have a high affinity antibody the concentration will
be somewherebetween 1 and 0.1 g/ml. When I do not have info on the
concentration of the antibody I always start with a 10-fold dilution series
and I test 4 different dilutions. With supernatants I use the dilution
series 1/1-1/10-1/100-1/1000. For a serum I use 1/10-1/100-1/1000-1/10000.
A negative control is always included. These first results will already
give you an indication in what region the optimal dilution will be. For
example 1/10 stains the whole section, 1/100 strong signal but still a lot
of background, 1/1000 signal and no background, 1/10000 hardly any signal
and no background. From this I conclude that the optimal dilution will be
between 1/100 and 1/1000. In the second experiment I test a 2-fold dilution
series with again 4 different dilutions: 1/200-1/400-1/800-1/1600.

When an indication about the dilution is given (e.g. by the manufacturer) I
start with the 2-fold dilution series as already described. E.g. when1/100
in immunofluorescence is mentioned in the package insert I test
1/50-1/100-1/200-1/400.

Hope this is of help.
Peter

========================================
Peter van de Plas
AURION
Costerweg 5
6702 AA Wageningen
The Netherlands
phone: (31)-317-497676
fax: (31)-317-415955
http://www.aurion.nl




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