Pgp9.5 glabrous skin staining

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! :wink:

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.