I have spent several months to optimize our nerve staining in human skin biopsies. We fix our biopsy 24h in Zamboni at 4 °C followed by 24h submersion by parafilm in 30% Sucrose at 4°C. Then they are cryosectioned at 50µm in Tissue tek at -20°C. I handle the coupes in a floating 48-well plate with a very small pencil with soft hairs. Then the staining protocol is as follows:

- 2x PBS wash of 5 min RT

- KMNO4 potassium permangate 15 min (oxidates pigment --> really cool to see)

- PBS 10 min

- Oxalic Acid 5% (Bleaches the melanin and other pigments)

- Blocking in 0.1% Tx, 4% NGS, in pbs for 1h at RT

- 1St AB 1:500 PGP9.5 (Abcam (ab108986)) in 1/2 block and 1/2 PBS overnight at 4°C

- then 2x wash in pbs

- 2nd AB at 1:300 (Usually Cy5 or something in the red spectre because green is often with autofluorescence)

... i get the lower nerve fibers in the picture, but we are really trying to get the full nerve plexus with the small nerve fibers that transpass the epidermis.

I am not able to find the small nerve fibers in my sections. I think/hope it may be related to the origin of material (stomach skin left over of surgery, which was then frozen for 2 months). I have never tried fresh material. But perhaps there are other factors involved. Please let me know what is your view on this :)

I included some pictures of my own results, as well as of a representable example study which shows great nerve plexus staining (Nolano et al. )

Thanks in advance,

Meagan