[Histonet] very specific background immunofluorescence in mouse embryo sections

[Andrea Hooper]

Do you have photos of the background? Glad to help with some more 
info ... Where is the background occuring, what organs and what cells 
are fluorescent? Could it be RBCs? How are you preparing the embryos 
prior to staining? Fixation, paraffin, frozen, post-fixation, 
blocking etc etc ...  Also are you using an particular type of pen or 
pencil on your slides which could be rubbing down into your sections? 
What agents are you using to coverslip? Do you use any sealant around 
your coverslips?

Andrea


At 1:02 PM -0400 6/11/08, Anna Elisse Beaudin wrote:
>Hello,
>
>   We've been doing some immunofluorescence in mouse embryos sections
>(E9-10), and have recently encountered a problem involving very specific
>immunofluorescent 'staining' in our negative controls.  the staining
>looks to be in specific cells in specific regions, and occurs in both
>our no-primary and no-secondary controls.   we are staining with a
>rabbit anti-caspase, followed by an alexafluor-conjugated donkey
>anti-rabbit. the background 'staining' if very bright and can be seen
>with several different filters.  has anyone else ever experienced
>anything like this?  we are stumped.  I have never had this kind of
>problem previously in other mouse tissues.  if anyone has any insight, I
>would greatly appreciate your input.
>
>Thanks in advance,
>
>Anna Beaudin
>Division of Nutritional Sciences
>Cornell University
>
>
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