[Histonet] mouse spleen autofluorescence quenching and other considerations

Gayle Callis gcallis <@t> montana.edu
Wed Aug 4 16:52:37 CDT 2004

   I   didn't   think   that   hydrogen   peroxide  is  meant  to  quench
   autofluorescence  (but  I could be misinformed) but rather it quenches
   endogenous peroxidase for immunohistochemistry peroxidase method.
    Sodium  borohydride  (blocks  free aldehydes and not without possible
   problems  on antigenicity loss per discussion by Jules Elias) has been
   used  to  quench  autofluorescence.   Some people have a terrible time
   getting  rid  of  autofluorescence and often never completely succeed.
   Autofluorescence can be worse aldehyde fixation.  As offered before, I
   have  a  review  of autofluorescence in tissues with GFP work in mind,
   but the discussion is very thorough, and pertains to nonGFP work also,
   it  includes  fixation  and  what in tissue autofluorescences.  I will
   send it to anyone via personal email.
   A  good  way  to  avoid autofluorescence in mouse spleen (an any other
   tissue)   is   cut   frozen   sections   and  use  either  acetone  or
   acetone/alcohol   fixative   (whichever   fixative   gives   the  best
   immunfluorescent   results   for  your  antibody)  avoid  NBF  or  PFA
   fixation.   Be  sure  to  have  one unstained section to see degree of
   autofluorescence and and if you use oil immersion, the oil must be PCB
   free and not contributing to autofluorescence.
   If  you  insist  on  FFPE  with  spleen,  you  can  cleverly  use  the
   autofluorescence   as   a  "counterstain",  so  to  speak,  have  your
   fluorophore be in contrast to the slight greenish-yellow, say an Alexa
   546  or  555  -  red  or  Rhodamine  or  even  Texas Red.  Jackson has
   secondary antibodies conjugated to a rhodamine RRX that is bright, and
   more  resistant  to photobleaching versus standard TRITC or Texas Red.
   The joy of Alexa dyes - very bright and resistant to photobleaching.
   A  side  note:    Molecular  Probes  has  "ready  to use" Prolong Gold
   antifade  mounting  media  (although  pricey and requires sealing with
   diluted  permanent  mounting media, not nail polish) - it sets up hard
   and  is  excellent  for keeping your fluorophores glowing longer.   We
   are delighted with this media, even with eGFP.

   At 02:02 PM 8/4/2004, you wrote:

     I  have  had  pretty  good  luck quenching autofluorescence in FFPE
     spleen with sodium borohydride 0.5 mg/ml in PBS pH 8.0 x 10 minutes
     x 3 changes.  It works better than H2O2 for me.

     Hi histonetters,
     Apologies  if  my  query  is  really  basic  but I am wanting to do
     antibody  staining on mouse spleen sections (paraffin-embedded) and
     am seeing a
     lot  of  autofluorescence,  which I presume is due to platelets. Is
     there any way
     around this problem?
     Helen Newbery,
     Medical Genetics Section,
     Molecular Medicine Centre,
     Western General Hospital,
     EH4 2XU.
     0131 651 1047
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     Sharon Cooperman             <scoop <@t> mail.nih.gov>
     NIH, NICHD, CBMB                     301.435-7735
     Building 18T, room 101               301.402-0078 fax
     Bethesda, MD 20892
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   Gayle Callis


   Research Histopathology Supervisor

   Veterinary Molecular Biology

   Montana State University - Bozeman

   PO Box 173610

   Bozeman MT 59717-3610

   406 994-6367 (lab with voice mail)

   406 994-4303 (FAX)


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