[Histonet] RE: Methyl Green

[C.M. van der Loos]

   Hi Mervi,

   The   brown-red  color  combination  you  mentioned  allows  a  simple
   hematoxilin  counterstain.  I  would advise you to work with a diluted
   hematoxilin   solution   and  keep  the  time  short.  Check  out  the
   counterstaining  result microscopically and if needed repeat.You don't
   want intensely blue nuclei as this will mask your double staining.

   If  you  really wish to counterstain with methylgreen soak your slides
   in  100  mM  Na-acetate  buffer  pH  5.2 for 15 min. Don't wash. Cover
   sections  for  5  min with 0.1% methylgreen (Sigma M6776) dissolved in
   the  acetate  buffer  as above. Wash briefly with with distilled water
   and  dry the  slides completely at a hot plate. Mount organically with
   VectaMount.  This procedure circumvents dehydration by alcohols, which
   isn't good for your red alk. phos. reaction product.

   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
   fax:    +31 20 6960389

   Date: Tue, 27 Sep 2005 14:19:05 -0700
   From: "Eeva, Mervi" <MEeva <@t> mednet.ucla.edu>
   Subject: [Histonet] Methyl Green
   To: histonet <@t> lists.utsouthwestern.edu
   Hi All,
   I tried Methyl Green counterstaining on my double stained
   immunohistochemistry    slides    and    didn't    get   any   visible
   counterstaining.
   Tissue was FFPE brain and otherwise I used mostly Vector reagents with
   DAB
   and AP-red detection.
   Any ideas what went wrong?
   - Mervi Eeva, UCLA