Clontech, Creative Diagnostics and Millipore all have anti-Cre Abs that they say work in WB and IHC. We also list some other possible sources. Just search here: http://www.linscottsdirectory.com/search/antibodies and follow "More Info" links for supplier's datasheet. Good luck.

You can perform a transient assay by transfecting some plasmid with loxP-stuffer-loxP cassette into your model system, and see whether the 'stuffer' fragment is looped out by the recombination of loxP x loxP, to test the gene product (CRE) biological function.

Thank you so much Tatyana! As we all know, sometimes companies sell antibodies that work in a very strange way, thus a personal experience is extremely useful!

Yuan-Yeu Yau, thank you for your suggestion concerning the control experiment. I did not mention in my question, but I would like to confirm expression of Cre recombinase in vivo, thus transfection is possible (in utero) but is a bit tricky.

Hi Pavel. Did you tested the millipore antibody? I am looking for a good one as well. 

Cheers,

Hi Carolina. I ordered this antibody: MAB3120 | Anti-Cre Recombinase Antibody, clone 2D8 from Millipore. It worked pretty well in ICC and IHC.

Hi Pavel, 

How well did the MAB3120 work for you? Would you mind maybe sharing the protocol you used? I'm currently trying to use it and we are not getting any real results. We're in the midst of troubleshooting it, actually. 

Hi Nicole. MAB3120 worked very well both in vivo (after in utero electroporation of the Cre-expressing plasmid) and in vitro in cultured neurons after a transient overexpression of the Cre-plasmid. Do you need a protocol for sections or for cultured cells?

Hi Pavel. I am also working on Cre-IHC in the brains of our Cre mouse line. I tried with Millipore EMD 69050 (originally from Novagen) and got very high background staining. Actually, I tried this with more diluted antibody, but it did not improve a lot.

With MAB3120, do you have any special treatment in your protocol? I would appreciate if you could share your protocol with me.

Thanks.

Hi Nicole and Kenzi. We use very standard protocols for IHC and ICC. You could find both protocols in, for example, Pellegrino et al., 2011 (PMID: 21486764).  Cre ab dilutions 1:500 and 1:1000 both worked well.

Hi guys, I am facing the same problem, I am using free-fro brain secitons, I have tried 2 different antibodies from cell signaling and Abcam, seems like very high backgroud, and the staining is not good at all. I am not sure if it is worth to try MAB3120. Could some one come and help

I'm having problem with mab3120 on IHC for OCT frozen section as well. There may be variation between lots. Did anyone try 69050-3, Novagen (or from Milipore) that works?

Hi Xuan, we have just recently published a paper where we used mab3120 IHC in OCT frozen sections ( PubMed PMID: 32064760). I agree that variation between lots could be a problem.

Hi,

For those who used the MAB3120, didn't you have problems when using a mouse host primary antibody into mouse sections? It seems that this antibody is the most used but I am wondering how you avoid unspecific binding in immunofluorescence staining.

Thank you!

Hi Sara,

As Pavel Uvarov mentioned, we recently published a paper (PubMed PMID: 32064760) where we used the MAB3120 Cre antibody in mouse embryos. We did have some issues with unspecific staining, but managed to get good results with a 1:1000 dilution in blocker (3% BSA, 0.3% Triton X, and 10% goat serum in PBS for) overnight at +4. Having a fresh stock of secondary Ab also helped. This was for E18.5 mouse embryos; the brains were not perfused, just postfixed in PFA for 4h.

Thank you Martina Mavrovic, I'll check your paper out!

Hi Martina Mavrovic , we're currently having the same issues with using MAB3120 on mouse brain tissues, but I'm interested in using the IHC protocol you used in your paper. Would you be able to share which secondary AB you used and at what concentration?

Hi, we used the Alexa Fluor goat-anti-mouse 488 at 1:500 in modified blocker solution (1% BSA, 0.3% Triton X, 10% goat serum in PBS) for 2h at room temperature. The sections were 40µm, free-floating.

I have now tried this antibody on section of the adult mouse spinal cord (perfused with PBS), and it worked well.

Thank you, excited to try it out - the images in your paper were great!

Hi Martina and Pavel, Congratulations for your publication,

I was not able to download your paper, could you please share full-length paper?

Hi Kambakam Sekhar , let me know your email so I can send it to you.

Hi Martina,

[email protected]