Re: [Histonet] Antibody dilutions and temperature

From:Rene J Buesa

  1- Abs + PBS: this is the neutral buffered environment the Ab should be diluted;
  2- Abs + PBS + albumen: this is the same environment but the addition of albumen will preserve the Ab better (for a longer time);
  3- Abs + PBS + albumen + Tween: the Tween, as a surfactant, will allow the Ab to "spread" better over the section and produce a better staining.
  You should dilute your Abs in PBS with aldumen (1%) + Tween 20 (0.05%) to obtain better results.
  Some instruments increase the incubation temperature to obtain quicker results, or the incubation can be done overnight at lower temperatures to allow for weaker dilutions and prevent evaporation. Temperature affects incubation spead according with Van't Hoff's laws (direct effect).
  When you have many experts in the same field with different opinions, either just 1 is right, or several have a "fraction of the truth" or all are wrong; make your pick!
  René J. wrote:
  Hello all,
I have noticed there are different diluents used for various antibodies; I 
have seen PBS, PBS plus albumin, and PBS/albumin/Tween. 
What is the difference between using one or the other of these? Also, how 
does incubation temperature play a role in the choice? Thanks for your 
answer, Tim :). Actually I would like to hear anyone's opinion since just 
about everyone here is an IHC or IF expert. Enjoy the weekend!

Melville B. Vaughan, Ph. D.
Assistant Professor
Department of Biology
University of Central Oklahoma
100 N. University Drive
Edmond, OK 73034
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