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 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.
@achamess
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.
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.
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.
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!
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!
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!