Jeremy, whats the protocol for your antigen retrieval?  Its unclear why you are getting EGR-1 to work and not c-FOS.  Its unlikely its the antiserum.  I would consider trying FRAS antibodies. The FRAS AS includes c-fos, fra-1, fra-2 and I think FOSb.  Its a nice approach because it actually measures IEGs that go are unregulated and down regulated so you can measure time course changes.  Check out work by Lerant, A.A. and Freeman, M.E. for details on FRAS. Good luck. 

Hi Michael,

Thanks for the fast response.

My antigen retrieval protocol is simply immersing the slides in pH 8.8 Tris-EDTA and then steaming in a steamer/rice cooker for ~10 minutes. Then I move onto a PBS wash, then blocking and normal staining.

When you use Fras antibodies, do you label for c-fos, fra-1, fra-2 and Fosb all at once? If so, do you have a recommended antibody (antibodies)?

I used to use an amazing c-fos antibody that was really strong and really specific. It was from Calbiochem. But recently they ran out of that lot and started sending out a new lot without telling anyone. The new lot didn't work at all and everyone I talked to said the same thing. After that we went on the search for an equivalent antibody, but so far as I can tell no one has found another one that was anywhere near as good. If you find one, let us all know.

Also, I do 60 min for my time course, which worked well for both c-fos and Egr1.

Hi Liz,

Thanks for the response. I've tried using this Ab http://www.emdmillipore.com/US/en/product/Anti-c-Fos-%28Ab-2%29-%284-17%29-Rabbit-pAb,EMD_BIO-PC05?bd=1 

I borrowed an aliquot from a neighboring lab and it worked well, so I decided to purchase. However, the new one I ordered did not perform/stain. You're right they definitely changed the lot and the new one is not as reliable/good as the previous it seems. 

Have you ever used something like pCREB or Egr1 in the hypothalamus specifically? 

I used Egr1 in the amygdala, and those sections included hypothalamus. I remember looking at the hypothalamus, but I didn't image it. I remember thinking that the Egr1 was less robust than c-fos and Arc (homemade antibody from Arnold Kriegstein's lab), so I didn't continue to use it.

I also made mRNA probes for c-fos, egr1 and arc, if you want those. The timeline for that is quite different though (~5 min).

Hi Liz,

I am trying pCREB right now, and will try another c-fos ab made in goat instead of rabbit from Santa Cruz. I will discuss potentially looking at the transcriptional level and using mRNA probes, although I am ultimately interested in protein. 

Chinmaya, I have multiple lots of sc-52 but can't seem to get it to stain in my tissue well with multiple anti-rabbit secondaries. 

Thanks again!

Let me know if it works. Santa Cruz ab's are hit or miss in my experience, with mostly misses. At least they give you a lot. Good luck. Email me if you want the mRNA probes.

Dear Jeremy,

It might be worth your while to look at the antibody database at Journal of Comparative Neurology (http://onlinelibrary.wiley.com/journal/10.1002/%28ISSN%291096-9861/homepage/jcn_antibody_database.htm).  While no database is perfect, but at least these passed the standards for use in a JCN publication.  (Their former Editor-in-Chief, Clifford Saper, was one of the first to raise the alarm about non-reproducibility of primary antibodies: http://onlinelibrary.wiley.com/doi/10.1002/cne.20839/full)

I see other anti-c-FOS antibodies listed other than sc-52, as well as other suppliers of other clones.  The benefit of the JCN database is it includes the reference for the journal article in which it was used, so you can check a recent publication and evaluate their staining and methods.  For instance, there is a 2012 publication that used Calbiochem's AB5, but no lot is listed, so it might be something they had for a while.  There is a totally different Abcam antibody (ab7963) whose use was published in 2011.

I do not work on the hypothalamus, so I checked out the pub cited by EMD/Millipore for neuronal labeling in hypothalamus, and the authors had to do what I consider a very sensitive detection technique:  free floating sections of fixed/frozen material, followed by 48 hr incubation in 1:1000 primary, followed by 24 hours with an HRP labeled secondary,  which was then detected with the Co-God method (glucose oxidase/nickel DAB).  To me, this suggests that there is very little c-FOS in these neurons, even though they needed a protocol for 40 um sections.   You may be able to get a similar label using fluorescence if you switch to green for c-FOS, and use the FITC-tyramide system.  Of course, this means switching your secondary antibody for the orexin to a different color.

I would also like to point out that when a commercially-available antibody doesn't work, I get in touch with the company to 1) let them know, and 2) get a replacement if necessary.  A reputable company should work with you to get their product to work, especially because the aliquot you borrowed DID work.

The EMDMillipore Ab that you tried and then ordered recognizes both v-FOS and c-FOS.  IF you are OK with that (I have not kept up on the FOS field at all), if worst comes to worst, there is also a hybridoma to almost the same synthetic peptide (residues 4-17 of human FOS) available from ATCC.  The hybridoma peptide had the serine at position 15 omitted, but the monoclonal  supposedly labeled in the same pattern.  I got good labeling (in goldfish!; not published), but I used the ABC reagent/DAB system.

Good Luck!

Jill

Hi Jill,

Thanks for the extensive and informative reply!

I am not familiar with tyramide amplification, but it may be something I turn to soon. I have seen plenty of articles showing cfos in orexin neurons covaries with vigilance state, e.g: http://www.jneurosci.org/content/21/5/1656.short

http://www.sciencedirect.com/science/article/pii/S0306452203003348#

And many others showing cfos immunoreactivity in the hypothalamus regardless of cell-type specificity. I may first try the hybridoma bank as their antibodies against the human antigen might work (the immunogen sequence is almost identical to the mouse's), and it's cheap.

I have contact Calbiochem to help with troubleshooting antibody problems with the ones I have from them, and Santa Cruz as well. It seems strange that I'm having such trouble getting good staining with such a widely used antibody. 

Dear Jeremy,

It is odd that you are having such problems, but antibodies are tricky beasts sometimes.  In fact, I just viewed a webinar from Science/AAAS on improved tissue profiling in mice (the first webinar on the bottom here: http://webinar.sciencemag.org/all-webinars/technology), and it is amazing how some simple step can be important.  Hopefully the different companies will send you the protocol they currently use to validate their staining, and tell you which tissues to use to do this.  Also, perhaps the others that have posted here would be willing to share their protocols?  Sometimes just following an exact protocol lets you get a handle on the problem. 

Since you have contacted the companies for technical help, I would suggest that you hold off on getting the hybridoma supernatant generated against the peptide with the serine deletion at position 15 unless that part of the peptide is not conserved in the mouse sequence.

Barring help from other sources, I do have a couple of additional suggestions.  Looking at the additional two papers you linked, they are similar to the EMD/Millipore reference in that they all use free-floating sections.  It could be that your 1 hour of drying at room temperature is changing the conformation of the antigen enough to prevent the primary antibody from binding well.  Also, none of the publications used any antigen retrieval techniques.  Free-floating sections get extensive washing in storage, so in many cases it is not needed.  Maybe you can try eliminating that step to see if helps the FOS staining.  Instead, just wash your slides longer at the beginning of the protocol--maybe 3 x 5 minutes, or even 3 x 10 minutes? (I don't know your section thickness.)

All three publications also use an amplification step of some sort in order to detect their primary antibody.  Now it is possible that they did this because they were using free-floating sections and the ABC reagent method is the easiest way to use DAB to get a permanent precipitate so that the sections can be mounted onto slides for clearing and coverslipping.  However, the fact that two out of three papers used more sensitive DAB mixtures, with added nickle or nickel-cobalt, still makes me think that you may need to do amplification to detect FOS in these particular neurons.  One simple thing you can try is to label with your secondary antibody for two hours at room temperature, rather than at 4 C.  While this might increase the background, it might increase the binding of the secondary enough to see a hint of a signal. 

For signal amplification, the easiest method to use is to substitute a biotinylated secondary for your fluorescently labeled secondary, and then to use a fluorescent streptavidin to create a fluorescent ABC complex.  Before you try the tyramide amplification, you will need to make sure you have a very clean secondary antibody of the type you need to go with the kit you buy, since it is a much fussier technique.  Both techniques will take more time to work out the reagent dilutions, so hopefully Tech Services will come through for you.

Good Luck!

Jill

Hi again Jeremy,

Since using mouse monoclonal antibodies on mouse tissue can present its own set of headaches, I went searching for my old protocol that I used on goldfish to see if it might have any useful hints.  I found that I had misinterpreted the information in our computer antibody database.  I did indeed test a hybridoma clone to that peptide, but we did not obtain it from ATCC.  A company had used that clone to make ascites; it was actually the company that notified us that they had been told that the antigen peptide was missing the residue at position 15.  So I don't know about the usefulness of the clone from ATCC, even though it has the same clone number.  The company unfortunately went through a couple of mergers several years ago, and no longer is in the antibody business. 

I apologize for my error, but I wanted to let you know before you ordered it and went through the work of setting up a new detection system.

Best of luck,

Jill 

Hi Jill,

Thanks again for the response. My sections are 25 uM mounted directly onto charged slides. I found that the antigen-retrieval step is crucial for improving my staining of many different antigens (iba1, GFAP, orexin, and Egr1) using the subsequent protocol. Perhaps you are right that simply more washing could allow me to skip this step.

See this paper for instance, where no antigen retrieval was used, but they use the same c-fos antibody I do (sc-52 from santa cruz) at a very low dilution (for Fluorescence I think), and get what seems like a good signal with a similar protocol (although these were free floating):

http://www.sciencedirect.com/science/article/pii/S0889159113005345

or this one

http://www.jneurosci.org/content/31/31/11376.short

I do my secondary incubation at room temp for 2 hours, maybe i made a typo in the original post.

Hi Jeremy,

Based on all of these references, I'm beginning to think that 30 um free-floating sections are the way to go to look for c-Fos in these neurons, regardless of antibody!

It's interesting that you need antigen-retrieval for anti-GFAP.  I have never needed to use it in my material, but then, retina and optic nerve have a lot of GFAP, and I use 10-12 um sections.

Good Luck--getting an immunolabeling protocol to work well is almost always the most time-consuming step.  If you decide that you need to try tyramide amplification, feel free to get in touch with me.  I have a pretty good protocol (see my anti-CNTF receptor paper in JCN).

Jill

Yes, I never thought that free-floating was necessary because I got good staining with a cfos antibody i borrowed from a neighboring lab (the discontinued PC38 from calbiochem). GFAP works without antigen retrieval, it just looks incredible with antigen retrieval.

I'm trying a few things now, but may try some amplification procedure soon if things dont work out. Thanks for all your help!

Hi Sean,

I was given a large lot of this anti-Egr1 from a neighboring lab that doesn't use it anymore. They had gotten it from a colleague several years ago, the name was A. Chaudhurri (it said on the box, and the PI can't seem to remember many more details except that he was from Canada.)

It was not purchased from a vendor though, sorry! It was made in rabbit.

 Hey Jeremy, did you ever get the cfos to work? A few labs here in psych are having so much trouble with the sc-52! 

Hi Sara,

Not fluorescently, unfortunately!

It works with DAB or SG substrate at 1:1000 in free floating sections.

I ended up doing double labeling with orexin with DAB, and cfos (sc-52) with SG substrate.

I don't know why it doesn't show up fluorescently! The only cfos Ab I was able to get to work fluorescently was PC-38 from calbiochem, which they do not sell anymore.

They said they have an equivalent one raised to the same epitope, PC-05...but that didn't work for me using an identical protocol...

So, I'd say if you can, switch to DAB/SG for double labeling if that's possible for you.

So frustrating. We are having a lot of trouble with it fluorescently as well, but I've had good luck with it using DAB. I'll have to consider that. Thanks!

Hi Jeremy, 

Have you tried using Alexa 488 for your cfos staining? I had a similar issue staining for a trancription factor recently. I was using alexa 546 and saw no expression, but using 488 showed great results. I believe the size of 488 is much smaller than 546 ot 594, maybe allowing better penetration into the nucleus for staining transcription factors. If you havent tried that, give it a shot. Just switching the fluorphores worked for me. Good luck!

Yes we got it to work with 594 secondary. The problem was using sections mounted on slides instead of free-floating. Worked great. See attached image (cFos in red, orexin-a in green)

Hi Jeremy,

What is the ultimate protocol that you now use for c Fos? Do you do antigen retrieval?

We also cannot get it working with fluorescence, only with DAB

Hi Nefeli,

I did not have to use antigen retrieval on 30 um cryostat sections (brains were from perfused (4% PFA) mice, that were post-fixed in PFA overnight followed by 30% sucrose for at least 3 days).

The protocol was:

Wash 5 min X 5 in 10mM PBS on shaker

Block for 1 hour in 2.5% Donkey Serum, 0.1% Triton-X100 in 10mM PBS

Incubate 24 h in primary antibody (rabbit anti-cFos sc-52; 1:200) in blocking solution at room temp on shaker.

Wash 5 min x 5 10 mM PBS

Incubate 2 hours in secondary antibody (Alexa-fluor 594 Donkey anti-Rabbit; 1:200) in blocking solution.

Wash 5 x 5 min 10mM PBS

Mount sections, spot dry and coverslip using VectaShield  media + DAPI

Thanks Jeremy, looks very straightforward!

So.. sc-52 ant c fos from Santa Cruz has been discontinued.

Any good alternatives?

I have a nice staining with that one, 1/200 overnight room temp, no antigen retrieval

https://www.cellsignal.com/products/primary-antibodies/c-fos-9f6-rabbit-mab/2250?N=4294956287&Ntt=rabbit+c-fos&fromPage=plp

The antibody that Arnauld suggested also worked for us on rat sections.

Thanks for the suggestion!

No worries!

Hi Arnauld,

Thank you for your share, I wonder which tissue do you used for your staining? I am trying brain slices.

Thank you!

free floating sections of rat or mouse brains

I am looking for an anti-cfos that is NOT raised in Rabbit (preferably not raised in mouse either) to use on rat free-floating 40 um brain sections.

Do you know of any good-working antibodies?

Nefeli - did you ever find an anti-cfos not raised in rabbit or mouse?

Yes, we use anti-cFos raised in Guinea Pig from Synaptic Systems (#226 004), with antigen retrieval (incubation of free-floating sections in citrate buffer for 30 minutes at 60 degrees).

It doesn't work as nice as the Rb anti-cFos from Cell Signaling #2250 (not sure if it is because of the secondary though), but it works quite well!

I keep seeing researchers talk about antigen retrieval, but it is not in my cfos protocol. Is this something that should be done for fixed sections? Thank you for providing information about the antibody!

Fixing often 'masks' antigens. So antigen retrieval 'unmasks' them.

In the case of cFos you still have signal without antigen retrieval but it is definitely stronger with it.

Be careful with temperatures, it might damage sections if it is too high.

Good luck!

Great, thank you. So just to be clear, you would do the antigen retrieval as the very first step, prior to applying blockers and antibody?

Jeremy C. Borniger Do you remember the company of the orexin antibody you used?