[Histonet] Bizarre Immunofluorescence problem in mouse brain sections

From:"Anna Elisse Beaudin"

Dear Histonet,

   I am having a bizarre problem with an immunofluorescence protocol that
I am really hoping someone can help me with.  here are the specs:

I'm doing immunofluorescence for BrdU in PFA-fixed mouse brain sections
that have been collected in PBS, mounted on slides, and allowed to dry 2+
days. Once dried, slides are processed for IHC as follows:
Antigen retrieval (20min in  .1M citrate buffer pH6.0, 95C)
2N HCl treatment
10% normal serum block followed by 10% casein block
rat primary incubation O/N at 4C
5min rinse with PBS, .1% triton X, + 2X3 min PBS
Secondary is a jackson Aexa488-conjugated goat anti-rat - 45 min at room temp
Rinses... coverslip

The problem I am encountering is the appearance of strange fiber-like
pieces on my tissue that are picking up the secondary.  at 40X they look
like segmented strings or fibers (almost like bacteria). I have no idea
where they're coming from.  I did NOT have this problem with regular HRP
development, and my secondary is brand new.  I have this same problem even
with a rat IgG control.  I am still getting good specific staining, I just
need to get rid of this stringy background.  Does anybody have any
suggestions/ideas about what this is and how I might get rid of it?  I
really appreciate your help.

Anna Beaudin
Division of Nutritional Sciences
Cornell University

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