[Histonet] RE: Confusing IHC results?

From:"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" 
   Subject: [Histonet] Confusing IHC results?
   To: "'histonet@lists.utsouthwestern.edu'"
   Hi everyone,
   I  just  completed  an  IHC  run  for  Calpain  1  (Santa Cruz), using
   1:800, 1:1000 and 1:2000. I also ran previous runs where the dilutions
   1:100,  1:200,  1:400 and 1:800. I am using a Universal secondary from
   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
   more  intense  and  has  more  non  specific staining than my slide at
   When  I  run  my  negative  at  any  of the concentrations to match my
   dilution, it is completely clean.
   What  is  going  on  here?  Has anyone ever experienced something like
   Primary  is a goat polyclonal. I have tried other secondaries such as,
   anti goat, horse anti goat, rab! bit anti
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