[Histonet] My Negative is now Positive ... IHC assistance needed?

GT Hebert emerald_lake77 <@t> yahoo.com
Thu Jun 2 16:14:06 CDT 2005

I recently worked up a new antibody for my lab.  It is a rabbit 
anti-mouse (antigen).  The antigen is found throughout the kidney and has been 
pinpointed by ISH to be located in the proximal tubules.  For the IHC, 
I have determined the best concentration for my antibody was 7 ug/ml.  
I used Jackson's ChromPure Rabbit IgG, whole molecule as a negative 
control at the same concentration, diluted in the same buffer as my 
primary.  I am also using the Envision system by DAKO.  
Lately I've noticed a lot of background coming up with my negative.  I 
re-ran a titre for my primary and had negatives at the corresponding 
concentrations.  To my surprise, I got a lot of background on the 
negative, and moreover, it appears to be decreasing as the dilutions get 
OK -- what's going on?  Have I contaminated my negative somehow??  I 
keep the glass jar it originally came in at 4 degree C.  When needed, I 
aliquot what I need under the Biosafety Cabinet (sterile conditions).  
If it has been contaminated, what has it been contaminated with and why 
is it giving me background?  How can I avoid this in the future (can it 
be aliquoted and frozen)?  Other suggestions will be very helpful.
My positive looks good.  Very clear staining and background only seen 
when Ab concentration is very high.  I need a clear negative though to 
support my primary Ab results.
Thank you all again for your help.

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