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Author Topic: False positive nuclear stain  (Read 575 times)

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Offline Fazzi

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False positive nuclear stain
« on: October 12, 2016, 04:47:22 AM »
Dear all,

I just started to use a Dako autostainer because I need to stain a lot of sections.
Testing a Ki-67staining on a multi-tissue section I noticed that the negative control (only antibody diluent for the primary) is not always negative.
Frequently the tissue shows a false positive nuclear staining that seems to be specific as not all nuclei stain positive. (See image)

Protocol:
Deparaffination in Xylene and rehydration to water.
HIER in Pt tank at high pH (EDTA pH 9) for 10 min. at 97 min. (Preheat 83 degr.C, 10 min. cooldown to 85 degr.C 10 min. Total time 30 min.)
Cooling down to room temp. in washbuffer.

Staining:
Rinse in Envision washbuffer containing Tween20.
Endogenous Enzyme Block: FLEX Peroxidase block, 5 min.
Rinse in washbuffer
Primary antibody: only antibody diluent, 20 min.
Rinse in washbuffer
Labelled Polymer: FLEX/HRP, 20 min.
Rinse in washbuffer
Rinse in washbuffer
Substrate-Chromogen: FLEX DAB+ Sub-Chromo, 10 min.
Rinse in washbuffer
Wash in water
Haematoxyline counterstain.
Wash in tapwater
Dehydration in Ethanol to Xylene
Coverslip

I tried all kind of things to figure out what is causing this nuclear stain which is variable in its intensity from one staining to the other.
We think it has something to do with the HIER treatment in combination with the labelled Polymer.
It definitely needs the labelled Polymer (which is Envision) to occur because without it the false positivity is gone. So this means that it canít be some endogenous enzyme activity either. Some tissues seems to be more involved in this than others. For instance larger hypochromatic nuclei are stained while tissue with smaller nuclei are not.

We hope someone recognizes this peculiar staining pattern and give us a clue of what is causing this.

Thanks,

Fazzi

False positive nuclear stain
« on: October 12, 2016, 04:47:22 AM »