Re: [Histonet] Bone marrow unspecific staining

From:Rene J Buesa

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  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 
- 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:
University of Helsinki Dept. Basic Veterinary Sciences

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Minusta se olisi erinomainen ajatus! 
- Gandhi


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