Hi Eva, I always remove all the OCT before I begin immunos even if there is only one big area to be encircled. After the section dries on the slide use a pair of fine tipped forceps to pull out the OCT. Pick up the dried OCT at the outer most edge and give a gentle pull the whole things comes off in one fluid motion leaving behind the sections untouched and unharmed stuck to the slide. Now you can use the pap pen to draw out your areas.

I hope this helps. Richa.

Hi Eva, besides taking out the excess of OCT, I have used sometimes colorless nail enamel (base coat) to encircle different tissues on a slide.

I also take off the outer OCT, but I have not yet gotten good IF results.

Is it bad to let the section/slide sit at room temperature while I do this? It could be dry at room temp for 15-20 minutes before rehydration, while I am removing OCT.

I have been using ImmEdge Hydrophobic Barrier Pen from Vector Labs instead of Abcam's PAP Pen. It's cheaper and and stronger than PAP pen for my IF and ICC. I hope this helps!

Thanks for all the good advices! Good to see that others had the same problem- and solved it for me:)