Re: [Histonet] My Negative is now Positive ... IHC assistance needed?
From what I understand from reading and through my own personal
experience, over time this Chrompure Rabbit IgG reagent from Jackson
gives background. I am not sure why. To avoid this I have heard
people say they aliquot and freeze it and only keep a working aliquot
at 4 deg C. I have this problem so much so with the Chrompure
Reagents that I am considering looking for another source for my
>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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