Re: [Histonet] Bone marrow unspecific staining

From:Andrea Hooper

Hi, Can you provide more details of your staining protocol? Without more info it is a little hard to guess what the 
problem may be. But on first glance these are my comments:

(1) Are your secondaries cross-adsorbed against whatever species you are working with? So for example if you 
are staining mouse tissue with rat  primaries, your secondary needs to be anti-rat IgG, no cross to mouse.

(2) BM is a tricky tissue with hard to quench endogenous peroxidases. If you are using HRP based protocols, you 
may need a special addendum protocol for peroxidase blocking.  [Doubtful though since you are using FFPE 

(3) What antigen are you staining for? Are you sure your antibody is "good"? Do you have a positive control which 
is not bone? What concentration are you using primaries and secondaries at? Did you titrate them?

(4) Finally, what were your tissues decalcified with?

I would suggest you run the following controls if you haven't done so already:
(a)  No primary, no secondary, no tertiary - only DAB - check for endogenous peroxidases
(b)  No primary, no secondary, only Strep-HRP and DAB - check for endogenous biotin
(c)  No primary, only secondary, tertiary, and DAB - check for secondary antibody non-specific binding
(d)  Isotype control of primary, then secondary, tertiary, and DAB - check for non-specific binding associated with 
isotype of primary

Good luck!

At 9:39 AM +0200 11/7/05, 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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