Antibodies for nociceptors that work well in skin?

We’re doing some skin staining with PGP9.5. Works great but I want to look for something more specific for C- and/or Ad-fibers. Can anyone recommend antibodies that work for targets like TRPV1, CGRP, Substance P, Nav1.7/8 or anything else that you’ve verified works well in skin?

@tberta @thicunha @sshiers

@achamess this is something we want to know as well. We have used Nav1.8 Cre/Tdtomato, but it will be fantastic to test Abs as well. Could you share your protocol for PGP9.5. We are trying to establish.

Yeah skin is tricky.
We are following the protocol of a mouse metabolic phenotypic core facility at UMich that does this often.

Here it is

Fresh mouse or rat:

  1. Remove footpad and fix (4 hours) in 2% zamboni’s fixative for 4-6 hours.
  2. Place skin in 30% sucrose in 1x PBS overnight.
  3. Embed tissue in OCT using scheme below.
  4. Cut 10-14 sections at 30um using scheme below and place into a single well of a 24 well plate.
  5. Follow IHC protocol using Ptroteintech rabbit anti-PGP9.5(14730-1-ap), 1:1000 followed by AlexaFluor 488 highly cross absorbed, 1:1000 (Molecular Probes). Incubate with DAPI, before applying coverslips with ProLong gold or platinum antifade kit with dapi (Molecular Probes)
  6. Block for 1-2 hours in blocking solution (500ul per well).
    7, Primary at RT for 1 hour, then in to cold room overnight (500ul per well).
  7. Rinse 3x1 hour in 1x PBS
  8. Secondaty at RT for 1 hour, then in to cold room overnight.
  9. Rinse 3x1 hour in 1x PBS.
  10. Incubate with 1x DAPI for 15 minutes.
  11. Rinse 3x10minutes each
  12. Coverslip using prolong without DAPI, one drop on each tissue piece.

Reagent 1:
Rinsing Solution
Reagents and Materials

  1. 0.3% Tx: 30ml 1% Tx in 70ml 1x PBS

Reagent 2:
Blocking Solution
Reagents and Materials
2) 5% BSA made in .3% triton tx100 in .1m PBS 7.4 pH
Weigh 2.5 grams of BSA and add 50ml 0.3% Tx in PBS

Reagent 3:
Primary antibody
Reagents and Materials
3) PGP antibody at 1/1000 made in 1% BSA in .3% triton tx100 in .1m PBS 7.4 pH
1% BSA in 0.3% Tx: 10ml 5% BSA and 40ml 0.3%Tx

Reagent 4:
Secondary antibody
Reagents and Materials
4) Molecular Probes anti-rabbit 488 highly cross absorbed secondary made in .3% triton tx 100 in .1M PBS 7.4 pH.

Reagent/Material Vendor Stock Number
Zamboni fix Newcomer Supply # 1459A
cryomold tissuetek
sucrose fisher
PGP 9.5 Proteintech 14730-1-ap
BSA sigma
Coverslip 1.5 fisher
slides Fisher
Prolong gold w/o dapi thermofisher P36931
Secondary 488 g anti Invitrogen A31620

Another version here

Thank @achamess. Did you perfuse the animals to collect before harvest the paw skin??

no perfusion. I remove paw first before perfusing.


  • Zamboni Fixative (Newcomers)

  • 30% sucrose in 1x PBS with sodium azide

  • Razor Blades

  • 1.5 mL tubes

  • 1.5 or 2 mL screw-top tubes


  1. Euthanize a mouse using isoflurane and cervical dislocation

  2. Use a razor blade to cut the dermal papillae in the middle of the mouse hindpaw

a. See video and image

b. After making the cuts, use scissors to finish removing (as in the video), being careful not

to pinch the epidermis. Handle the tissue from the deeper dermal tissue below.

  1. Drop the tissue using forceps (holding from the deeper face) into the Zamboni fixative.

  2. Let fix for 2 hours at RT

  3. After 2 hours, remove the Zamboni fixative. Add 30% sucrose/PBS to wash the tissue until no

yellow stain remains (2-3x).

a. Collect the Zamboni fixative in a 50 ml conical for waste. Dispose in PFA waste.

  1. Then add 30% sucrose/pbs and let incubate overnight at 4oC

  2. On the morning of 2022-08-29, transfer the tissues to screw-top tubes and replace the 30%

sucrose with fresh 30% sucrose.

a. To transfer, dump out the skin into a well of a 12-well plate. Using clean forceps, pick up

the skin from the deep side and transfer into new screw top tube. Note: After every 3 mice, use a new razor blade. It needs to be sharp.

Thank you again. We will try. Please let us know when you find good Abs for additional markers.