[Histonet] Bone marrow unspecific staining

[Pamela Marcum]

Paraformaldehyde is just freshly made formaldehyde solution from powder in 
a buffer.  It should not make a difference in the staining or 
background.  As this is a bovine sample in origin you may need to change 
your blocking serum.  You do not give the type of serum so here I can not 
really make a suggestion.  Animal tissues often requires making adjustments 
not suggested in standard protocols.

Pamela A Marcum
UPENN Vet School
Kennett Square, PA
610-925-6278

At 09:50 AM 11/8/2005, Rene J Buesa wrote:
>Although I cannot be absolutely sure, for me it seems that perhaps the 
>problem may reside in the paraformaldehyde fixation. Do you have to use 
>it? Could you use another fixative in another parallel sample just to try 
>to find out?
>Just a thoght!
>Rene J.
>
>Mikael Niku <mikael.niku <@t> helsinki.fi> wrote:
>Andrea and Rene, thanks for your comments. I'm sorry for being a bit
>unspecific with my unspecificity question :)
>
>Some more details on the problem:
>
>- Seems to be caused by unspecific antibody binding, but not a specific
>property of this primary ab
>- Primary antibody works very well with other tissues
>- Also other primaries (and secondary alone) show unspecific staining
>with BM
>- Not caused by endogenous biotin, peroxidase, or autofluorescence (done
>enough controls)
>
>And some more details on the protocol:
>
>- Bovine BM tissue samples decalcified with EDTA, or cell samples
>embedded in agar
>- Paraformaldehyde fixation
>- HIER (glycine-HCl pH 3) + Tween-20 permeabilization + mild protease
>digestion
>- For peroxidase-based protocol, biotin block (tried also peroxidase
>block, doesn't help)
>- Serum block
>- Rabbit primary antibody (overnight +4C)
>- Sheep secondary antibody (biotinylated or fluorescent)
>- For biotinylated secondary, tyramide amplification & DAB reaction,
>hematoxylin counterstain
>- For fluorescent secondary, Sudan Black B for autofluorescence suppression
>- Embedding with Faramount
>
>--
>////////////////////////////////////////////////////////////
>
>Mikael Niku URL: www.helsinki.fi/~mniku/
>University of Helsinki Dept. Basic Veterinary Sciences
>
>- Mitäkö mieltä olen länsimaisesta sivistyksestä?
>Minusta se olisi erinomainen ajatus!
>- Gandhi
>
>////////////////////////////////////////////////////////////
>
>
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