[Histonet] background!!

[RCHIOVETTI <@t> aol.com]

In a message dated 5/11/2005 10:59:30 AM US Mountain Standard Time, 
pex0220 <@t> yahoo.com.cn writes:

> I think that it isnot autofluorescence due to no background in negative 
> sections, I am really sad because I can not find an effective method to reduce 
> background. Any suggestions will be helpful! I am looking forward for your 
> help!
> 

Guofeng,

I don't know whether this will help or not, but it is worth a try.  I used to 
use this technique to solve background problems for immunoelectron 
microscopy:

After both the primary and secondary antibody steps, rinse a few times in 
normal PBS, then rinse a few more times in PBS with a very high concentration of 
salt.  I used 5 Normal PBS, meaning the saline contained 5 times the amount of 
salt it should have.

After you rinse with the concentrated salt solution, be sure to wash a few 
times in regular PBS, then with PBS containing the blocker.  You have to return 
the slide to a normal concentration of saline before proceeding to the next 
steps.

Normal saline is about 150 mM, or 0.9% NaCl by weight.  Try making up a 
saline solution that contains about ** 4.5% ** NaCl by weight (750 mM).

This worked very well (when nothing else would) at the EM level, so there's a 
chance it will work at the light level as well.

This tip comes from a physical biochemistry lab where I was a postdoc.  We 
did a lot of column chromatography, and we used high salt washes to clean up our 
columns.  

The salt probably alters the conformations of the antibodies and probes so 
much that only the specifically interacting ones are able to hang on in the 
concentrated salt.

Good luck.  Let us know if this solves the problem.

Bob

Robert (Bob) Chiovetti, Ph.D.
Independent Consultant for The Science, Technology and Industrial Sectors
132 North Elster Drive
Tucson, AZ 85710-3212 USA
Tel./Fax 520-546-4986
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