[Histonet] RE: Histonet@lists.utsouthwestern.edu

[C.M. van der Loos]

   Hi Gayle and others,

   Just some remarks on the methanol-issue from my perspective:

   Basically  you  are  right  that  methanol  is nasty stuff for many CD
   markers.  Using  methanol on human cryostat sections has a deleterious
   effect  on  far  most  antigens  (yes, I am aware that the mouse-world
   thinks  different on this). However, with FFPE sections that have been
   through  alcohol-steps  several  times a methanol step doesn't seem to
   matter  that  much  anymore.  Once  an antigen or epitope has survived
   embedding,   methanol   will   not   do   any   harm.   All   my  FFPE
   sections developed for peroxidase activity are blocked with methanol +
   0.3%  peroxide  for 20 min at RT (Streefkerk, 1972 in JHC) without any
   problem.  Some  people  claim  there are exceptions here, but so far I
   haven't seen real comparison experiments proving this.

   Blocking of endogenous peroxidase activity with methanol+0.3% peroxide
   is  far  more  effective than PBS+03.% peroxide+0.1% sodium azide. The
   latter  just blocks erythrocyte pseudoperoxidase but just "weaken" the
   endogenous peroxide activity in neutrophils.

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


   Date: Wed, 11 Jan 2006 08:42:11 -0700
   From: Gayle Callis <gcallis <@t> montana.edu>
   Subject: [Histonet] Methanol and CD markers
   To: Lori Richey <lrichey <@t> u.washington.edu>,
   Histonet <@t> lists.utsouthwestern.edu
   Just a note here. Methanol is known to create problems with CD marker
   immunohistochemistry  and  that  is  why many use hydrogen peroxide in
   buffer
   instead  of this solvent.   This point was brought up in Jules Elias's
   book
   on  immunohistochemistry  and  also  on  Histonet  in  the  past. (see
   archives).
   At 04:02 PM 1/10/2006, you wrote:
   >We  use  both CD4 and CD8 on FFPE sections.  We had problems with the
   CD4
   >until  we changed our quenching step to 0.5% H2O2 in MEOH for 10 min.
   The
   >pretreatment for both is 15 min microwave in pH 8 EDTA.
   >
   >Thomas Pier wrote:
   >
   >>Hello,
   >>I am looking for Reinhard von Wasielewski.  I recently saw a post
   >>stating tha! t he had