Re: [Histonet] Bone marrow unspecific staining

From:"Andrea T. Hooper"

Bone is FAMOUS for being tricky to work with so 
everything must be done with patience :) Here are 
my new batch of thoughts based on your problems:

- Do you have a picture of the background??? Is it specific or diffuse?
- Is your rabbit primary an antiserum or a affini-pure antibody?
- Is your sheep anti-rabbit cross adsorbed against cow tissue?
- What species was your anti-rabbit secondary made in?
- Bone marrow is filled with cells of 
hematopoietic origin which have a lot of 
endogenous peroxidases as well as Fc receptors 
which may bind to your primary antibody (or 
secondary for that matter). This is why isotype 
controls and a serum block is so necessary. So, 
what serum have you been using for blocking?
- Why are you using tyramide? Is this necessary for your antigen?
- Have you titrated the primary at different 
dilutions and at different times for incubation?

I doubt that the PFA is the problem as I 
routinely work with PFA fixed murine BM samples 
and don't have any issues but how long are you 
fixing for?
You do the fixation *after* EDTA decal correct?

>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 
>- For fluorescent secondary, Sudan Black B for autofluorescence suppression
>- Embedding with Faramount
>  Mikael Niku             URL:
>  University of Helsinki  Dept. Basic Veterinary Sciences
>  - Mitäkö mieltä olen länsimaisesta sivistyksestä?
>  Minusta se olisi erinomainen 
>                                          - Gandhi


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