[Histonet] RE: Double staining of 2 antibodies with 2 different

[C.M. van der Loos]

   Patrick,

   With  this type of trouble there are usually no solutions. However, in
   your case there is perhaps one:

   You  may  perform  your  double staining sequentially starting without
   antigen retrieval. Incubate with the ubiquitin antibody followed by an
   appropriate    polymer/HRP.    Visualize    HRP    activity with   DAB
   (obligatory!).  Then  perform  HIER with citrate and continue with the
   HLA-DR  antibody  etc. ending with AP activity in red (allowing a weak
   hematoxilin  counterstain).  In general, a brown-red color combination
   is  not  suitable  to  observe  real  co-localization by mixed colors.
   However,  you  said  that  you are looking for structures near to each
   other, so this will be no problem for you.

   Hope this helps!

   Good luck,

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


   Date: Fri, 19 Jan 2007 11:20:44 -0800
   From: "Patrick Laurie" <plaurie <@t> benaroyaresearch.org>
   Subject: [Histonet] Double staining of 2 antibodies with 2 different
   retrieval methods
   To: "histonet <@t> lists.utsouthwestern.edu"
   <histonet <@t> lists.utsouthwestern.edu>
   Hello Histonet,
   I am trying to do some double labeling of ubiquitin and HLA-DR LN3
   antibody on human spinal cord. My PI wants to be able to see both the
   ubiquitinization of the motor neurons with patients that had ALS, as
   well  as  seeing  the proliferation and activation of microglia on the
   same
   slide, near the same cell.  But, I have found that the ubiquitin I am
   using, (Chemicon's Monoclonal ubiquitin antibody) requires no antigen
   retrieval (AR), while the HLA-DR LN3 antibody (monoclonal from MP
   biomedical) works best with a 30 min citrate retrieval.  The citrate
   retrieval with the Ubiquitin creates too much background staining,
   blocking reagent! s are pow