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

- - emerald_lake77 <@t> yahoo.com
Thu Jun 2 15:45:54 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 higher.  
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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