Have you tried adding some detergent in your washing buffer?  0.3% Tween 20
or 0.1% Triton-X 100 + 2.5% BSA in your primary antibody incubation buffer
as well as in the first 2 washes immediately after your primary and
secondary antibodies will help  to reduce alot background.  But you need to
wash off detergent well before your peroxidase step.  Also if time is not
too strict, overnight incubation of primary antibody (in buffer with
blockers and detergent) at 4 degree C at higher dilution can give you more
specific staining.
Hope this will help!
> -----Original Message-----
> From:	Kim Kusser [SMTP:kkusser@trudeauinstitute.org]
> Sent:	Friday, August 18, 2000 12:35 PM
> To:	histonet@pathology.swmed.edu
> Subject:	PNA staining
> 
> Hi all,
> 
> I've been trying to reduce the non-specific background staining I get when
> I use PNA (biotinylated). I block with 5% BSA (more than enough), and
> Avidin/Biotin block (vector kit). I've titrated the dilution as high as I
> can without losing the specific staining that I want. Problem is...PNA is
> one sticky lectin. Either that and/or it has other things it likes to
> stick to besides the germinal centers...like epithelium.
> 
> BTW, I stain frozen mouse sections...spleen, NALT, Peyer's patches, Lymph
> nodes and lung.
> 
> All tips and tricks appreciated
> 
> Kim