Methods and Techniques Discussion > Immunohistochemistry (IHC)

Patchy Staining: can PAP pens interfere with stainings?

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EJN:
We're doing a MAG staining on paraffin brain samples; several slides from different blocks from different patients. And we have noticed spoty inconsistencies in the intensity of the DAB over the tissue after development that aren't attributable to difference in MAG. They are either drop-shaped regions or whole portions of the slide where the DAB is clearly lighter than in neighboring areas. The intensity doesn't progressively change but is rather separated by a clearly visible line of darker to lighter DAB regions on the tissue.

I'm thinking maybe an antigen retrieval issue, but my coworker seems convinced it could be an issue with the PAP pen leaching into the tissue or disassociating during wash steps into the buffer and then reattaching to the tissue when removing the slides from the wash buffer. Has anyone witnessed anything like this before? What are the odds this could be the PAP pen? We are setting the barrier several  mm away from the tissue on the sections...   

CanuckPhD:
I have not had this issue with any of the brands of PAP pens that I have used. You just have to give it a few seconds to dry after application before you add the solution and you are okay. I usually apply it before the block, so after fixation and quenching.

What you are describing could be due to the AR. Are you ensuring that there are no hot and cool spots in your container? You have to mix the solution often, especially if you are using a home type microwave for AR. We finally bit the bullet and bought a dedicated lab microwave that has constant stirring to avoid these issue.

BJessica2:
Are you washing adequately between steps? including tween-20 in your wash buffer, may help...

I have never had a problem with PAP pens.

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