My experience is that 1) fixation is important and if using paraformaldehyde, you cannot heat the solution to dissolve it above 53˚C or it eats the tissue when it turns into formic acid. 2) I like 48 hrs for primary better than 24 hrs if the antibodies are fussy. I do not add anything (no normal serum, no blockers of peroxidase (unless perfusion is not very good), I make up my own DAB solutions do not buy pre-made solutions since the company does not tell you what buffers they use. I dilute the secondary to between 1:600 and 1:1200; I dilute the ABC reagents too (see our protocols or the pdf of the paper), I would always include another antibody made in the same species so that I know that everything other than the primary is working well in that tissue. I store my sections in antifreeze solution and do not try to stain it for at least one week after cutting (it does something magic that I do not understand, but freshly cut tissue usually does not work as well as stored tissue. I do not think that if a Cre antibody is working, it will benefit from antigen retrieval. That process is not as useful for freely floating tissue as it is for paraffin/frozen sections. Anyone wanting our protocols should email me and I can send them

It can work beautifully. See this paper

Wolfe A, Divall S, Singh SP, Nikrodhanond AA, Baria AT, Le WW, Hoffman GE, Radovick S. Temporal and spatial regulation of CRE recombinase expression in gonadotrophin-releasing hormone neurones in the mouse. J Neuroendocrinol. 2008; 20(7):909-16. PMID: 18445125. The antibody was from Covance and was used at a dilution of 1:2,000,000! Yes 2 million. At higher concentrations the antibody did not work as well. Please see my article on how to titrate antibodies as it will give you an approach that we feel is best for establishing the correct primary antibody concentration and a clue as to how to then pick the right method for seeing that antibody.

Hoffman, G.E., Sita L.V. and Le, WW. Importance of titrating antibodies. Current Protocols in Neuroscience Chapter 2 Unit 2.12, 2008. PMID: 18972376

I haven't tried newer lots, so if Aaron bought some more recently and it is not good, I cannot comment, but sometimes very avid antibodies will look as though they do not work at high concentrations but if diluted substantially work quite well.

That is my hope!

Thanks guys! I've used the 69050-3 Rabbit anti-Cre from Millipore and also the MAB3120 also from Millipore but without results.

I've shifted to doing cryostat sections now since I can do antigen-retrieval on them but was hoping to keep that as my last resort since it destroys some of the epitopes for another protein I want to co-localise with.

@Gloria: If this doesn't work. I'll try using the antibody you suggest and check.

Hi Gloria,

I tried using the PRB-106P rabbit anti-cre mentioned in your publication (I hope this is the one?).

I've tried combinations from 1:50 all the way to 1:1,000,000 but don't see satisfactory labelling in any case. I've used free-floating sections cut on a vibratome as well as cryostat cut sections (for 1:50-1:500 dilutions) but no encouraging results.

Unfortunately, I've not been able to access the second publication you've cited above. I was wondering if you could send me a detailed protocol along with a copy of the titration you've used.

I just need to get this done asap as I need to submit my paper and thesis subsequently.

Any help would be greatly appreciated.

here is our standard protocol If you email me at [email protected] I will attach the titrating article

I'll give this a try

I've done mostly as your protocol states (except for using PBS instead of KPBS) and did the 48hour incubation as well. I just checked and found that this antibody is recommended to be used at 1:50-1:100 and is a replacement for the older one which has been discontinued.

I'm at my wits end with what to do and might go down the in-situ route if this doesn't work soon.

Thanks for taking time out to help me though.

My experience is that 1) fixation is important and if using paraformaldehyde, you cannot heat the solution to dissolve it above 53˚C or it eats the tissue when it turns into formic acid. 2) I like 48 hrs for primary better than 24 hrs if the antibodies are fussy. I do not add anything (no normal serum, no blockers of peroxidase (unless perfusion is not very good), I make up my own DAB solutions do not buy pre-made solutions since the company does not tell you what buffers they use. I dilute the secondary to between 1:600 and 1:1200; I dilute the ABC reagents too (see our protocols or the pdf of the paper), I would always include another antibody made in the same species so that I know that everything other than the primary is working well in that tissue. I store my sections in antifreeze solution and do not try to stain it for at least one week after cutting (it does something magic that I do not understand, but freshly cut tissue usually does not work as well as stored tissue. I do not think that if a Cre antibody is working, it will benefit from antigen retrieval. That process is not as useful for freely floating tissue as it is for paraffin/frozen sections. Anyone wanting our protocols should email me and I can send them

if you want to email me some photos of what the staining looks like, I might be able to help trouble-shoot the methods better (just reduce their size to 150dpi and no bigger than 4x6 inches. jpegs are good

I have found that the only way to know if an antibody has activity is to first screen it in a positive control series of sections using Elite ABC immunoperoxidase methods. This is key in that with a fluorescence based method, you lose sensitivity by a factor of approximately 100-200 fold. As the titration method paper shows, with the same conditions (primary antibody and tissue), detection will be minimal with direct-tagged fluorophore on the secondary antibody and so you lose the ability to tell if low activity is present. If it is, then the TSA amplification for fluorescence will work but direct tagged secondaries may not. Sometimes use of amplification works well in chromogen-based methods giving 10-50 (or more) times the sensitivity but it requires careful adjustments of antibody concentrations or it maxes out.

It would be beneficial to get a cre transgenic line (or someone's sections from one known to work well) with Cre under a very active promoter so that you can have a 'positive control' Some of those mice with low activity promoters can be much harder to detect. If you know first that the antibodies would detect the protein if present in sufficient amounts, you'll be way ahead of the game to learn if your system works. I've only looked at the GnRH Cre line and someone brought testicular sections with Cre under one of the Sertoli cell promoters and both worked well. I can try and send you some sections that I know worked (and in fact had generalized expression to test with your sections, but you will need to treat the tissue with sodium borohydride). Once we get some system that has strong expression we try and keep a large number of test sections to verify other systems when needed. (In Edinburgh how complicated will it be to receive fixed brain sections from the states that should be cool but not frozen?)

Hi Navneet! Did you finally get to work Cre staining in brain sections? I´m considering to order the MAB3120 antibody from millipore and I was wondering if you found a better antibody to detect Cre recombinase in brain sections.

Thanks!

Hi Martin, did you try the antibody you mention after all? If yes on what kind of tissue and how where your results? 

Thank you in advance

Zisis

Hi Navneet,

I was wondering, did you manage to find a good anti-Cre antibody that worked in youyr free-floating sections? I am looking for a good option to use in my experiments.

Thanks!

Hi Inês - I don't know if you're still looking for an option, but our lab has had some success using this antibody in mice: Novagen rabbit: 69050 (https://www.emdmillipore.com/US/en/product/Anti-Cre-Antibody,EMD_BIO-69050)

We've also used the following antibody to some success: LSBio mouse: LS-C79918 (https://www.lsbio.com/antibodies/anti-cre-recombinase-antibody-aa77-343-ihc-icc-if-immunofluorescence-wb-western-elisa-ls-c79918/80544)

you really should use ABC elite technology and Nickel DAB as the chromogen.  The way you listed is going to be 100 times less able to detect the Cre. Free floating sections work wonderfully as our paper indicates.

Dear Ian, 

I would like to give it a try with the antibody that you suggested...Maybe is asking too much but could you send me the protocol that you used? my email is [email protected]

Thanks a lot

There's no information there about Cre antibodies yet, but this database I just came across might turn out to be very useful: http://pabmabs.com/wordpress/

Perhaps the ones of you which obtained some sucess with the Cre antibody can add a review there.

Cheers

Ines

Rabbit anti-Cre (69050) worked really well for me on perfused rat sections,

at 1:1000 and secondary goat anti-rabbit 488 from Abcam

HI, Navneet

Now I am facing the same problom as you did 5 years ago, I tried several antibodies, but no one worked. I use mouse brain free-fro section. How did you solve the problem in the end. And probably I need to try the Rabbit anti-Cre (69050) as Nefeli recomended.

Looking froward to your reply

Thanks

I have learned a wonderful simple trick to prescreen antibodies. Take the antibody and using a micro dot blot (