[Histonet] Hazy background during IF

[Adam .]

Seasons Greeting from the laboratory:

I've been having a somewhat puzzling issue with my IFs lately. I stain
mostly fixed, decalcified mouse bones, and recently, my sections have this
strange haze over them. It's not consistently but it does crop up in maybe
10% of sections. It looks like a dense fog settled over my sections,
especially in the red/594/TxRed channel, often obscuring the signal. It
sometimes does this it the green channel and rarely in the DAPI channel. If
I carefully strip off the coverslip and soak the sections in PBS for a few
minutes, and then re-mount, the problem goes away. I'm guessing it's a
problem in the mounting somewhere.

Here is my protocol:
Sections are circled with a PAP pen. All liquids are dispensed using a 200
uL pipetter onto it
Finish staining
Wash 3 x 5' in TBS-T
Add a drop of Prolong Gold Antifade (I previously used Vectashield and had
the same problem)
Put on coverslip
Dab corners of coverslip with clear nailpolish
Let sit overnight for mounting media to cure

Any ideas what could be going on?

Thanks,
Adam