Thanks @sshiers. I knew you’d have some great insights to share. I hope the next antibody works out for you. Keep us posted.
My first round using AR (heated citrate pH 6.0) didn’t reveal any of the CGRP+ fibers that I was looking for in my human lung samples: still a lot of background, or nothing much of anything with a 1:16000 dilution I did to make sure that all that background wasn’t obscuring something positive. I’m fairly certain that I have TUJ-1 working (no AR necessary) and have seen nerve fibers in my samples, and some of them should be CGRP+ …but it also could be that I’m just not seeing any biologic positivity. Keep me updated and I’ll keep working on it too!!
Hello everyone! I know it has been a while since the latest post in this thread, but I was wondering if anyone has had success with this CGRP antibody or other markers for FFPE. I would greatly appreciate any protocols or advice!
@sowderme Can you tell us more about your application? Mouse or human tissue? What structures are your trying to visualize?
I am trying to perform IF staining of innervation in the bone using mouse FFPE tissue. We would like to ultimately visualize sympathetic and sensory nerves in the bone. I have seen this antibody used before in bone, but it was with frozen sections and not FFPE. I have tried CGRP staining in the skin and bone using citrate antigen retrieval, overnight incubation with the antibody at 4C (1:200-1:5000), and fluorescent secondary antibodies with no success.
Which antibody are you using?
I’ve had success with the Sigma antibody in PFA-fixed mouse tissues (DRG, SC)
https://www.sigmaaldrich.com/catalog/product/sigma/c8198?lang=en®ion=US
Skin is variable. I’ve never gotten great skin staining.
Yes, I have been using the Sigma antibody. Would you mind sharing your protocol for DRG, SC staining? I haven’t tried staining these sections previously.
Sure.
From this paper:
Animals were deeply anesthetized with isoflurane and transcardially perfused with 4% paraformaldehyde. After perfusion, lumbar DRG (L3–L5) and spinal cord were removed and postfixed in the same fixative for 2 h at 4°C. Then, the tissues were cryopreserved in 30% sucrose/PBS solution for at least 24 h. All tissues were mounted in optimal cutting temperature (OCT) medium (Tissue-Tek) or PBS (free-floating sections) and cryosectioned using a cryostat (Leica). For immunofluorescence and in situ hybridization, DRG sections were cut at 12 μm and thaw-mounted onto Superfrost Plus slides (VWR), and spinal cord sections were cut at 14 μm and thaw-mounted.
For immunostaining of DRG, sections were blocked in a solution containing 1% BSA and 0.4% Triton X-100 for 1 h at room temperature (RT). After blocking, the sections were incubated overnight with primary antibodies diluted in 1% BSA with 0.2% Triton X-100 at 4°C. After washing 3 times in PBS for 5 min at RT, sections were incubated with the appropriate secondary antibody for 1 h at RT followed by 3 washes in PBS for 5 min. Before mounting, some sections were counterstained with DAPI and fluorescent Nissl stain (NeuroTrace 640/660, Thermo Fisher Scientific). Slides were then mounted in Prolong Gold (Life Technologies) and allowed to dry overnight at room temperature.
And here are the images:
Also , see this convo on Twitter:
Good luck! Please let us know how it goes. I’ve heard bone is challenging.
Great, thank you! I just want to clarify that you used the same staining protocol for frozen and FFPE sections?
The protocol is for PFA-fixed. This is all I’ve done. I never did fresh. FFPE (with formalin) should be comparable.
@achamess glad to see this thread is still running… not as glad to see that we’re all still looking for a good human CGRP ab!
I still haven’t gotten my CGRP to work, and more recently I’ve attempted a VIP stain on my human airway biopsies. It also didn’t “work”: I think i have a positive stain, but no fibers are staining! Similar to CGRP, my VIP stain shows up in glands and perhaps some other types of cells. Could this be because the neuropeptide was released by the nerve and I’m seeing it having been taken up into cells?
For those of you who have stained peripheral nerves with CGRP: do you get nice nerve fiber-looking structures, like what you might see with a pan-neuronal marker?
@mckleroy Check out this paper looking at CGRP fibers in human skin
This is the antibody. The paper cites it incorrectly (Inkstar), but I’m pretty sure this is it.
https://www.immunostar.com/shop/antibody-catalog/cgrp-calcitonin-gene-related-peptide-antibody/
This paper also cites it, and has some of the other neuropeptide targets you’re looking for
1 Like
Hey friends,
I tried the Immunostar CGRP in mouse DRG and nerve. Looks great!
Will post pics later. Now, does it work in human? Idk. Someone let us know if you try.
looks like we both had the same thoughts… I ordered and used the Immunostar one as well. Works great on human!
That is on human DRG and spinal cord. I havent tried nerve
1 Like
@sshiers Thanks for letting us know! This is why I love this forum. Everybody doesn’t have to reinvent the wheel. Looks like we’ve figured out the human CGRP staining problem.
Here is our new paper using the Immunostar CGRP antibody on human DRG and spinal cord.
1 Like
Nice paper! And thanks for posting here. Your CGRP staining looks great. I’m got my hands on some human skin tissue (both frozen and in paraffin) to use as a positive control and will give that a shot soon.
Separate question from your paper: do you think NaV1.7 (or 1.8, I suppose) or peripherin would stain the axons of every sensory nerve? I’m looking now for a pan-sensory neuronal marker, and it seems like those 3 might be good options. Thoughts?
Thank you! 
None of those will work. In human, Nav1.7 is in most of the neurons, but not all (the very large ones 100um+ are negative). Nav1.8 at least at the mRNA level is only in the nociceptors (co-positive with CGRP/P2X3R neurons). Peripherin protein seems to be in most if not all sensory neurons, but its most abundant in small-diameter neurons. At least in rodent, its thought to be a small-diameter neuron marker but it doesn’t look that way in human. To be safe, I’d go with Beta-3 tubulin.