Note that there is precise immunolabelling at the external aspect of the tissue, which becomes weaker  towards the centre of the tissue ( zonal positivity)

This is due to inappropriate primary fixation.

This tissue was fixed in “PFA” for 24 hrs at 4C with no agitation.

A mistake: For FFPWS one needs to fix at RT (  NOT slow fixation)WITH agitation  and Fix:tissue 25:1 ( use a rocker-roller).

Formalin fixation is an ongoing process but, needs to be facilitated for FFPWS, unlike perfuse-fixation for cryosectioning.

Why? because inadequate fixation allows the alcohol in the processing to act as a fixative as well as a dehydrant to a greater degree than required.

Imho.

I never use “PFA”. I make up my Formalin using a ~30% Formalin solution.