There are many variables in the preparative of a biological sample to the cryostat.

For example, you can perform the first sections and then fix them.

Alternatively you can use in polylysine-coated slides or alternatively slide with electric charge specially in trade for these uses.

Have you tried snap-freezing your sample in liquid nitrogen instead of fixing it in PFA? literally, just snap-freeze, then mount onto your chuck and section.....no faffing. I also recommend trying poly-L losing coated slides as these will help to "bond" the section to the slide. I usually cut my sections at 7um. The hole-y appearance you describe sounds like freezing damage which sometimes can be caused by the sample defrosting and freezing again slowly - usually happens if you take too long mounting the sample onto the chuck and sometimes even storage at -80 instead of liquid nitrogen can cause artefactual holes in your samples (I've learnt this the hard way!!)

Helen x

Thank you for your replies, Fabio and Helen. I will try snap-freezing some ovaries tomorrow and will section onto some gelatin subbed slides. Is there a fix you have found more effective for cytosolic proteins?

The fixative you choose to use does depend somewhat on your antibody......when I previously did some immune for beta caterin I had much more luck with an ice cold acetone fix instead of a formalin fix. You might find you need to trial a few different options as you customise your protocol.

Helen x

When you formalin-fixed, then you need to run a wash of equal duration to that of the fixation.

Fabio