Re: [Histonet] Bone marrow unspecific staining

From:Mikael Niku

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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