[Histonet] RE: mouse spleen confocal

[C.M. van der Loos]

   Dear Scott,

   You  didn't  tell us what detection system was used. Your story sounds
   reminds   me   to   our   first  experiments  with  streptavidin-based
   detection on   mouse   spleen  when  we  were  confronted  with  heavy
   endogenous   biotin!   For   staining  mouse  spleen,  either  avoid a
   streptavidin-based detection   system   or   try  to  block endogenous
   biotin as Gayle suggested.

   Please  realize that if the above is true and/or Gayle's is right with
   her  suggestion  concerning  the  adsorption  of the anti-rat reagent,
   the signal  you observe is no autofluorescence but "unwanted" specific
   fluorescence!

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


   From: Scott Parker [[1]mailto:scott9379 <@t> gmail.com]
   Sent: Tuesday, April 18, 2006 9:05 AM
   To: histonet
   Subject: [Histonet] mouse spleen confocal
   Dear all,
   Can anybody help me with some antibody staining I have been doing on
   mouse
   spleen? We are trying to detect CD8 in 5 um sections using CD8b.2
   (ly-3.2) (
   53-5.8) with secondary Alexa Fluor 633 from mol. probes. Unfortunately
   all
   we see is vast amounts of auto-fluorescence making antibody detection
   impossible. We really need to get this sorted because ultimatley we'd
   like
   to  have  GFP  and  two  different  secondaries (labelling 3 things in
   total).
   Our protocol is basically:
   Spleens are removed to OCT and frozen in isopentane, sectioned (5 um)
   and
   stored at -20.
   When ready for use slides are warmed to room temp and fixed in -20 C
   acetone
   for 3 mins then re-hydrated in PBS for 5 mins.
   Secti! ons are a

References

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