[Histonet] RE: Confusing IHC results?

[C.M. van der Loos]

   Hello Dusco,

   When  antibodies  are applied at a far too high concentration staining
   will  get  weaker.  This  is  called  the  "prozone  effect".  I would
   suggest to  go  up  with your dilutions like: 1:2000 - 5000 - 10,000 -
   20,000  -  50,000.  At  least  until  the specific staining disappears
   again.   Lately,  I  came  across  the  same situation  and  ended  up
   unexpectedly at 1:200,000-500,000!

   Stupid  remark perhaps, but did you take care that your blocking serum
   isn't normal goat since your secondary is anti-goat?

   As far as the reward: I keep it to the drink!

   Chris van der Loos, PhD
   Dept. of Pathology
   Academical Medical Center M2-230
   Meibergdreef 9
   NL-1105 AZ Amsterdam
   The Netherlands

   phone:  +31 20 5665631
   fax:    +31 20 6960389


   Date: Tue, 19 Jul 2005 17:11:13 -0400
   From: "Trajkovic, Dusko" <dusko.trajkovic <@t> pfizer.com>
   Subject: [Histonet] Confusing IHC results?
   To: "'histonet <@t> lists.utsouthwestern.edu'"
   <histonet <@t> lists.utsouthwestern.edu>
   Hi everyone,
   I  just  completed  an  IHC  run  for  Calpain  1  (Santa Cruz), using
   dilutions
   1:800, 1:1000 and 1:2000. I also ran previous runs where the dilutions
   were
   1:100,  1:200,  1:400 and 1:800. I am using a Universal secondary from
   vector
   at 1:200 on all of the above slides.
   My staining results do not vary at all. As a matter of fact, my 1:2000
   looks
   more  intense  and  has  more  non  specific staining than my slide at
   1:100.
   When  I  run  my  negative  at  any  of the concentrations to match my
   primary
   dilution, it is completely clean.
   What  is  going  on  here?  Has anyone ever experienced something like
   this.
   Primary  is a goat polyclonal. I have tried other secondaries such as,
   bovine
   anti goat, horse anti goat, rab! bit anti