[Histonet] Re: Bone marrow unspecific staining

From:"Johnson, Teri"

Thanks for the additional information.  Only you know what you've tried
and haven't, so I'll give you some things to consider:

~you indicate the antibodies are working well in "other tissues."  And
you are currently using "Bovine BM tissue samples decalcified with EDTA,
or cell samples 
embedded in agar".  You may have to use a different pretreatment
protocol to get these same antibodies to work in decalcified bone and
whole cell preparations.  It could be that the pretreatments you are
using in these samples require a different concentration of primary or
secondary antibody.

~Try pH 6 or pH 8 HIER solutions with and without enzyme and see what

~Any time I see non-specific nuclear staining, I immediately suspect the
retrieval method combined with antibody concentration.  Just because you
haven't seen this in other tissues, doesn't mean you won't see it due to
differences in specimen types (cells v. tissues) and handling (decal)
prior to staining. Do you see any specific staining at all in either?
If so, take the dilutions of the primary and secondary antibodies down
to reduce nonspecific background. If you're not seeing any specific
signal at all, I wouldn't bother trying to abolish background staining,
especially if the answer lies in further antibody dilution.

~If you are seeing this effect only in the tyramide amplified samples,
again check the concentration of your antibodies.  It could be you need
to dilute them out further.  Or try using a streptavidin conjugated
enzyme or fluorophore and see if you get signal with less background and
go from there.

~You may never get some antibodies to stain well in decalcified
aldehyde-fixed bone marrow.  We had this issue with some markers and
ended up using non-fixed, non-decalcified frozen sections using the
Cryo-Jane tape transfer system.  This takes time and practice to use on
these difficult samples, but it may be another option.

~if you've got cells to stain, why not just fix and stain the cell
cultures rather than paraffin processing them?  Some antibodies work
better in ICC protocols (permeabilization only and no digestion or
retrieval) than for paraffin-embedded IHC protocols.

Good luck!

Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64133

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