Hi all, could anyone recommend a good protocol for pgp9.5 staining of mouse foot pad skin? I get some dermal pgp9.5 staining, but never the small nerve endings that should enervate the epidermis. I have seen many different protocols - could really use some advice from someone that has it up and running! thanks, v.
Hi @vanja. Good question. I too see these beautiful PGP9.5 images and wonder how they get them. @zhzhj131421 has done some beautiful PGP9.5 staining in our lab. He can chime in. I also recently saw a paper that had solid skin staining and I noticed that they used a different fixation procedure than PFA.
- Arcourt, A., Gorham, L., Dhandapani, R., Prato, V., Taberner, F.J., Wende, H., Gangadharan, V., Birchmeier, C., Heppenstall, P.A., and Lechner, S.G. (2017). Touch Receptor-Derived Sensory Information Alleviates Acute Pain Signaling and Fine-Tunes Nociceptive Reflex Coordination. Neuron 93, 179–193.
L3-L5 DRGs were dissected in ice cooled PBS, fixed with 4% PFA for 30 min at 4°C and incubated over night in 30% sucrose at 4°C. DRGs were then embedded in Tissue-Tek O.C.T compound and cut into 20μm cryo-sections. After drying, sections were incubated in 50mM Glycine for 20min, washed twice with PBST (0.2%), blocked with PBST (0.2%) + 10% horse serum + 1% BSA and then incubated with primary antibodies for 1h at room temperature. Primary antibodies were diluted in PBST (0.2 %) + 10% horse serum. Sections were then washed four times with PBST (0.2%), subsequently incubated with secondary antibodies for 1h at RT, washed with PBST four times, dried and mounted with fluorogel (Fluoprobes). Skin samples were fixed with methanol/acetone (1:1) for 30 min at -20°C, washed four times and incubated in 30% sucrose at 4°C overnight. Samples wereembedded in Tissue-Tek, frozen with liquid nitrogen and cut into 50μm cryo-sections. After drying, sections were incubated in 50mM Glycine for 45min, washed twice with PBST (0.2%), blocked 1h with PBST (0.2%) + 10% horse serum + 1% BSA and then incubated with primary antibodies overnight at 4°C. Primary antibodies were diluted in PBST (0.2 %) + 10% horse serum + 1% BSA. Sections were then washed several times with PBST (0.2%), subsequently incubated for 4 hours with secondary antibodies in PBST (0.2 %) + 10% horse serum + 1% BSA at room temperature, washed with PBST (0.2%) several times, dried and mounted with fluorogel (Fluoprobes). Lumbar spinal cords were dissected, fixed with 4% PFA for 4h at 4°C and incubated o/n in 30% sucrose. Tissue samples were embedded in Tissue-Tek O.C.T compound, frozen in liquid nitrogen and cut into 30μm sections. Spinal cord sections were stained as described for c-Fos stainings (see below). Image analysis and stack assembly of confocal images was performed off-line with ImageJ.
thanks @achamess ! I saw a few protocols, and this is the first with the methanol/acetone fix. what seems to be really different from what I have tried before is the horse serum. since we already have all our sections fixed and cut we will try this first. if we don’t get anything different we will test our the meth/acetone procedure.
@vanja Good luck! Please let us know how it goes. Something else is the use of glycine. We don’t do that routinely, but I know the purpose is to reduce autofluorescence. Skin is very autofluorescent, especially in the green channel. So that might be how they get good signal to noise. Also, check out that paper for the exact antibody they used and dilutions.
great tips! we will try next week, I can post before and afters! 
Hey @achamess we are just now imaging the new stainings - we are using alexa 555 instead of 488, we have included the glycine step and are using goat serum (10%) for blocking instead of 1% BSA. we are visualizing the small fibers in teh epidermis and it looks great. can you recommend some (easy?/automated?/best?) ways to quantify? thanks a million for your suggestions!
Hi @vanja. That’s fantastic! I’m so happy. Thanks for sharing. Which PGP9.5 ab did you use and what dilution? Glad to hear that even with PFA(?) fixation, the staining looks good. Maybe it’s the glycine and high serum buffer that does it. Alexa555 is definitely helping too since that’ll reduce the background. I haven’t done much skin quantification, but I think people usually do the density of fibers/unit area.
@tberta: do you have any thoughts about easy/fast skin quant?
You are right Alex. Most of us use density (number) of fibers/unit area, but some also use total length of fibers/unit area.
I think both are valid measurements. To note, the length is time consuming but also more sensitive to changes.
We use Zytomed 516-3344 at 1:200 as recommended. the skin (flank and foot pad) was only postfixed in 4% para/PBS overnight, 30% sucrose to cryoprotect and frozen on a block of dry ice in OCT.
Hi. Just wanted to chime in with my own results. I didn’t do PGP9.5 (I will…), but I did CGRP and GFP (transgenic). We got the best skin staining we’ve ever seen (in our hands at least). Thanks to @mny3 for doing the heavy lifting on this. We did the protocol as outlined in the Arcourt 2017 paper. Not sure which part was most important. It could be the fixation, the glycine, the heavy blocking. @vanja’s results with PFA fixed tissue suggests it might be the glycine and blocking that are most critical. In any case, now I know what I’ll be doing for those textbook quality skin staining images.
A member of our lab recently published a paper including PGP9.5 staining in the skin of the plantar hindpaw. Her images turned out pretty nice, so you can see her methods in the paper here:
The antibody choice is important here - we flailed around with an Ab from Fitzgerald for a long time before switching to the Dako one, which worked beautifully on the first try.
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Thanks so much @liz
I was having a hard time getting the ab from the UK company. I’ll look in the Dako one.
Hi all. Circling back to this discussion.
For the quantification of nerve fibers, I know many people do it manually adjusting the Z-focus and count by eye.
If you want to count using images, you need a Z-stack and confocal (I think). Can anyone share a protocol or a specific reference on how to do this?
Can anyone please recommend a good PGP9.5 antibody for staining skin sections? It seems like the one from Dako was discontinued…
thanks
Looks great. I will definitely try this antibody.
Do you use it on floating or directly-mounted skin sections?
If it works on directly-mounted sections that would be great since I already have some sections mounted and stored
Here it the protocol that was used
Free-floating, but I think slide-mounted could work too.
Fresh Mouse or Rat Footpad Removal & IHC:
Removal & Fixing:
- From euthanized subject, remove hind foot.
- Slice underneath papillae moving down from the digits towards “palm”, then slice at a perpendicular angle freeing papillae from the foot, observe procedure under microscope for more accurate removal.
- Place removed foot pad section in Newcomers supply 2% Zamboni fixative for 2-4 hours.
- Place footpad section into 30% sucrose in 1x PBS overnight.
- Embed tissue in OCT cryomold, place in -80°C freezer.
Sectioning: - Remove OCT cyromold from freezer.
- Adhere OCT block to cryostat mount using OCT.
- At 30µm, slice into the frozen footpad section, remove a test slice and stain using toluidine blue. Observe under microscope.
- Once the nerve trunks begin to be visible in the test slice, cut 10-14 sections off the footpad and place into a single well of a 24 well plate with 1x PBS (place well plate into cold room if keeping overnight).
Immunohistochemistry: - Carefully remove 1x PBS from well plate without removing footpad sections.
- Add created blocking solution into wells (~500 ml per well) and block for 1-2 hours. (See 2nd page for solution formula).
- Add Primary antibody solution at room temperature, ~500 ml per well let sit for 1 hour, then store in cold room overnight. (See 2nd page for solution formula).
- Remove primary solution, and rinse footpad sections 3x in 1x PBS for 15-20 minutes each rinse(minimum).
- Add Secondary antibody solution at room temperature, let sit for 1 hour then place in cold room overnight. (See 2nd page for solution formula).
- Remove secondary solution, and rinse footpad sections 3x in 1x PBS for 15-20 minutes.
- Incubate footpad sections with DAPI for 15 minutes.
- Rinse sections in 1x PBS 3 times for ~10 minutes each.
- Coverslip sections with ProLong gold, or platinum antifade kit without DAPI, ~1 drop on each piece of tissue.
Reagent formulas
• Blocking solution: 5% BSA in 0.3% triton
a. Mix 30 ml of 1% triton tx100 stock with 70 ml of 1x PBS to create 0.3% triton solution.
b. Weigh out 2.5 grams of BSA and dissolve into 50 ml of 0.3% triton solution.
• Primary antibody solution: Ptroteintech rabbit anti-PGP9.5 1:1000 solution
a. Create a 1% BSA solution via mixing 10 ml of the 5% BSA solution in 40 ml of 0.3% triton.
b. Add PGP antibody at a 1:1000 ratio into 1% BSA solution (1 µl PGP for every 1 ml of 1% BSA).
• Secondary antibody solution: Molecular Probes anti-rabbit 488
a. Mix 488 secondary antibody in a 1:2000 ratio with a 1% BSA solution (1 µl 488 per 1 ml triton).
• Reagent/Material Vendor Stock Number
• Zamboni Fix Newcomer supply #1459A
• Cyromold Tissuetek N/A
• Sucrose Fisher N/A
• PGP 9.5 Proteintech #14730-1-ap
• BSA Sigma N/A
• Coverslip 1.5 Fisher N/A
• Slides Fisher N/A
• Secondary Alexafluor 488 g antibody Thermofisher A-21206
• ProLong Gold w/o DAPI Thermofisher P36931
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Thank you! That’s very helpful!