Re: [Histonet] Bone marrow unspecific staining
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
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 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: www.helsinki.fi/~mniku/
> University of Helsinki Dept. Basic Veterinary Sciences
> - Mitäkö mieltä olen länsimaisesta sivistyksestä?
> Minusta se olisi erinomainen
> - Gandhi
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