[Histonet] RE: weak antibody signal

[C.M. van der Loos]

   Renee,

   I  have  three detection/visualization  procedures for you to consider
   for testing (no. 3 is based on the assumption that you are intended to
   stain FFPE sections; no. 1/2 can be used for cryo's too):
    1. Most  simple is just to repeat what you did so far but ending with
       Vector  NovaRed  kit  instead  of DAB. This very sensitive kit has
       helped us in similar situations a couple of times here.
    2. We  have  been pretty successful lately with the detection of goat
       antibodies using a 2-step method ending with a polymer. The second
       step  was  a  rabbit anti-goat Ig (Dako) 1:5000, 30 min RT and the
       final step EnVision+ anti-rabbit. DAB+ was used for chromogen.
    3. As you said the CSA kit might be a good solution too. In case of a
       goat  antibody  your second step should be rabbit anti-goat Ig/HRP
       (Dako)  1:100,  15  min,  RT.  Then  continue  with  Amplification
       reagent,  etc.  Please  realize  that  the  reaction product after
       tyramide  amplification  is somewhat more diffusely localized than
       with other IHC detection procedures.

   In  all  three  cases  you  should perform new dilution series of your
   primary.

   Good luck!

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

   phone:  +31 20 5665631


   Date: Tue, 22 Nov 2005 10:44:27 -0600
   From: "Till, Renee" <TillRenee <@t> uams.edu>
   Subject: [Histonet] weak antibody signal
   To: histonet <@t> lists.utsouthwestern.edu
   We have a BTEB1 goat polyclonal antibody that is staining very weakly
   using a Vectastain kit and DAB. I personally like to order my
   secondaries and streptavidin separate, that way I can get F(ab')2
   fragements or what have you, if I want. I was going to suggest trying
   this, as it is has worked better for me on some stains than the Vector
   kits. Is there anything else that could be done to boost the signal? I
   had  thought  of  CSA  kits and the like, but from what I can tell the
   Dako
   kit at least isn't for goat. Would it work better perhaps using
   fluorescence instead of DAB?
   Renee' Till, HT
   Arkansas Childrens Nutrition Center
   1120 Marshall
   Little Rock, AR