I want to stain DRG neurons and skin for CGRP. I’ve tried the goat anti-CGRP from Abcam and sometimes its good, others not so much. Abcam (36001)
I’ve seen the anti-CGRP (rabbit) from Peninsula referenced too, but it’s not produced for me. Does anyone have a really good CGRP antibody?
I’d love to get great staining like this amazing figure form Zylka (2005)
Zylka, M.J., Rice, F.L., and Anderson, D.J. (2005). Topographically Distinct Epidermal Nociceptive Circuits Revealed by Axonal Tracers Targeted to Mrgprd. Neuron 45, 17–25.
In this paper, they use Zamboni fixative and the Peninsula antibody, but I haven’t had such luck. Any tips?
I recently used the rb CGRP antibody from Sigma (C8198) 1:10,000 dilution. It worked very very well in the mouse joint and surrounding tissue, with several different fixative, embedding, and decalcification procedures, and with and without antigen retrieval. It worked equally well in TBS or PBS and with Tween 20, Triton X-100, or both. However, I have not tried it with fluorescence. I was using DAB staining with a histofine kit secondary antibody (Nicheri Bioscience, Japan) which may be more sensitive (but you do lose any stoichiometric relationship between the staining and antigen). Be sure to add 1% BSA to the primary antibody or you will get high background. My only complaint is that it did not last many freeze thaw cycles.
Good luck & if you do use it with fluorescence let me know if it works!
@Taves Hi Sarah! Thanks for joining the forum. So good to hear from you.
Thanks for the info on the CGRP antibody. Looks pretty sweet and sounds like it’s robust in many conditions. I’ll give it a try. Pretty pricey (~$500), but if it gets results, it’s worth it.
I’ll post results when/if I use it.
I just received some new antibody last week. Someone stuck it in the -80 prior to aliquoting it but hopefully it is fine. The last antibody I had stored at -20 but it was a frost free freezer (we don’t have one that does not defrost) thus the repeated freeze thaw cycles. You are welcome to take some. As I am using it at 1:10,000 so I think it is more likely to go bad before it runs out.
I am at UNC now but am working from home today. I technically on vacation starting Monday but do not leave town until early Thursday morning. Just let me know if you’d like to come by and pick some up.
PC205L | Anti-Calcitonin Gene Related Peptide (CGRP) Rabbit pAb (Millipore) is the antibody we used in the lab for many years. The staining is pretty good with this antibody, but you need to use high concentration between 1/200 to 1/500 in Zamboni fixed tissues.
Thanks Temo. I’ll look into this too. I’ll also try side by side with the one @taves mentioned. I did use Zamboni fixative to prepare the skin. It does make a difference. While we’re on the topic, how about PGP9.5? Do you have recommendations for that?
The Sigma CGRP antibody that @Taves recommended is superb. Very little background, strong staining, appropriate localization. I used at 1:1000 to 1:2000 dilution on DRG neurons and got excellent images. Best I’ve ever produced. Thanks to @taves for the rec and sharing some antibody. I’ll post images later for you all to evaluate for yourselves.
Have you also tried using this antibody in skin sections?
Just curious because we have been having issues with the CGRP staining in the skin.
Yup! It’s beautiful there too. We just did it today. This is the only antibody for CGRP that doesn’t give me lots of nonspecific backgound.
Hey I’m Ashley, I am in Francie’s lab and have been working on the CGRP staining. I was wondering if you could share your skin staining protocol? I got one from the Zylka lab and used the antibodies they suggested, but I have not been able to get good staining of CGRP in neurons in the skin.
Here is what we do.
Tissue Fixation and Sectioning
- Transcardially perfuse with 4% PFA (3 mins, 25 ml)
- Post-fix the paw (I take the whole paw, some people take just the skin) for 1-2 hours on ice.
- Cryoprotect in 30% sucrose/PBS.
- Cut out the glabrous skin and underlying muscle (down tot he tendon).
- Freeze the section in OCT and cut 20 um sections. Thaw mount onto superfrost plus slides.
- Hydrate in PBS for 5 mins
- Block with blocking buffer (1% BSA, 0.4% Triton-X, PBS) for 1 hour at RT
- Incubate with primary antibody (1:1000 CGRP ab, 1% BSA, 0.2% triton x) overnight.
- Wash 3x in PBS
- Stain with secondary antibody (anti-Rabbit Cy3, 1:1000) for 30 mins at RT
- Wash 3x.
- Counterstain with DAPI and mount.
@zhzhj131421 may chime in. He did the staining today. But the above is my very basic IHC protocol. Nothing different than normal. I tried the Zylka protocol from the paper cited above, with Zamboni fixative, and I didn’t see much. But I haven’t used the good Sigma CGRP ab that I use now. Maybe the Zamboni would make it even better? I think the antibody really matters. Also use Red or Far-red for secondary. Skin has so much autofluorescence, green is really hard to make out.
An update: the Sigma CGRP ab works really nicely in the cord too. 1:500, 14 um mounted slice
@areynolds @tberta @Taves
Hi Sarah, what was the dilution on your secondary?
1:1000 ant-rabbit Cy5 conjugated.
Have you tried the Sigma CGRP ab on human? Datasheet says it only reacts with rat…
I haven’t. I’m sorry. Worth a shot, or trying one that is reported to work on human.
I am currently trying to get it to work on paraffin-embedded sections from human lung or trachea; I’ve gotten a beta3 tubulin (R&D TUJ-1) to work with a Vector labs peroxidase secondary setup, but can’t get the CGRP to work yet. Have tried 1:1000, 1:4k, 1:8k and seem to have a lot of background. Planning on giving an antigen retrieval process a shot; didn’t need it for TUJ-1 so was hoping to avoid it. Will keep you posted. Thanks @achamess for the help in this thread! And if anyone has success in human with their CGRP, let me know!
Thanks for joining the forum. Let me ping our resident IHC expert, @sshiers. good luck!
I’ve tried 4 different CGRP antibodies with and without pepsin AR on hDRG, including the recommended Sigma ab, but no luck so far. The label is either non-specific or negative. I ordered one that is supposed to see human CGRP (and is published to work in human), so I will keep you updated when I know more!