T/S through spinal cord and DRGs.

Note intensest positivity at floor plate region

Positivity in muscularis of oesophagus and vagus nerves

Papilla of kidney shows, what I assume to be, positive  collecting ducts ( baso-lateral positivity in the simple cuboidal lining epithelium).

However, the epithelia at the proximal portion of the papilla appear to be negative for NKCC1 (  lower left of image).

Explanatory comments are most welcome.

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.

Using an “enhanced” DAB solution, I can use this primary at 1/2K and higher, rather than the 1/1K that  I use for my std stABC-PX-DAB.

One may use micropolymer kits but, in my limited experience, it is the ” enhanced” DAB in these kits that gives the enhanced sensitivity. Using a micropolymer secondary merely saves time…but, not necessarily costs 😉

My std HIER solution is Citric acid pH6.4. This ab does not work unless I use TRIS pH10 HIER solution. Also, this ab, using my std homemade DAB is diluted 1/50 for optimal staining. When using SteadyPlus, I can dilute to 1/400.

Surrounding tissue is mouse, therefore negative. Arrowhead points to mouse hair follicle.

Strong membraneous positivity.

Occasional nuclei are also positive.