[Histonet] Re:human-on-human IHC

From:"Andrea T. Hooper"

Hi Jamie and Brett,

I can confirm that this is a very good approach and works well for 
human on human staining, however it is only useful for those antigens 
which are fairly robustly expressed as some sensitivity of the 
protocol gets lost when precomplexing.

If possible, a much more sensitive way to go is to buy a labeling kit 
from Molecular Probes and label the antibody - with biotin, FITC etc. 
You can be as creative as you imagine ... It only takes about 1.5-2 
hours (much of which is an incubation step) and makes life infinitely 
easier. The large number of kits they sell are suitable for all 
different amounts of starting protein and a wide variety of 

Good luck!

At 1:42 PM -0400 5/1/06, Jamie E Erickson wrote:
>Hi Brett,
>                      I haven't done exactly what your talking about but
>this may help. I'm sure your secondary anti-human will be a problem.
>When I did some crossreactivity studies in various species with a
>humanized antibody I used a protocol that was a Modification of the method
>of Tuson, Fung, Hierck and their colleagues (Tuson et al. 1990; Fung et
>al. 1992; Hierck et al. 1994) that involved mixing the primary and
>secondary together overnight at 4 degrees C in a tube.  the next day  I
>added gamma globulin (human) to bind unbound secondary then this mixture
>was applied to the section like a primary  antibody. I had good results in
>various species with little background  but did not have human tissue to
>try it out on.  It just one way to get around the secondary nonspecific
>If you would like more information contact me I think I have the protocol.
>Jamie Erickson
>Sr. Research Associate
>Department: DSMP
>Abbott Bioresearch Center
>100 Research Drive
>Worcester, MA 01605-4341
>FAX: 508-793-4895
>e-mail: jamie.erickson@abbott.com


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