Re: [Histonet] Bone marrow unspecific staining

From:Rene J Buesa

In the description of your steps I don't see you blocking the endogenous peroxidase; do you do that step (5 minutes with 3% hydrogen peroxide after HIER)?
Rene J.

Mikael Niku  wrote:
Dear Histonetters,

I'm trying to do some immunos with bone marrow sections, but have 
problems with unspecific staining.
It seems a large proportion of the BM cells is unspecifically binding 
any antibody, including my secondaries.

These are paraffin-embedded sections of paraformaldehyde-fixed material, 
either decalcified tissue samples or agar-embedded cells.
I have tried to improve blocking, for example by increasing the serum 
concentration in the pretreatments, and by adding serum to the antibody 
solutions, but it doesn't help at all.
I have also tried different secondaries (biotinylated and fluorescent) 
but the results are always the same.

I need to do HIER in an acid buffer for my most important primary in 
this project, but it doesn't seem to be the cause of the problem.

Any ideas, anyone?

With best regards,


Mikael Niku URL:
University of Helsinki Dept. Basic Veterinary Sciences

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