Hello, Noemie.

Most of people use enough extraction buffer (i.e., either HPEM or PBS buffer+0.02% Triton X-100) for 30~60 min when tissue is large (they are numerous layers of cells and tough ECM & cell-cell contacts) to permeate cell and nuclear membranes. Xylene is not commonly used in the Cryostat section due to its immiscible properties to H2O. Remaining excess Xylene may be present deep in the nucleus. Then the antibody solution containing H2O would not have access to the nucleus. Find out cryostat section & IF procedure in detail without using Xylene (and smelly gas and highly toxic).

As you already know well, many TFs like c-fos are found in both cytoplasm and the nucleus, c-Jun is necessary for nuclear translocation of the complexes with c-fos. So c-Jun is an indispensable partner in that process. If your tissue shows cytoplasmic distribution of c-fos (and the histological process was O.K.), that is not impossible. The cells of your interest were not activated at the time of death (link). https://www.researchgate.net/post/SOX2_staining_in_the_cytoplasm

With a new protocol without Xylene in your adjusted procedure, have another go.

Best wishes.

Thanks a lot Mr Lee for your answer. Usually I used Xylene at the very end of the IF process just before covering the slices with the mounting medium. In my actual lab we don´t have Xylene so I didn´t dehydrate the slices, I just mounted them directly after washing the antibody solution. I was not sure if this can impact the preservation of the slices or not.. But good to know that it is not indispensable.

For the c-fos labeling, I thought that what I see is c-fos protein which had migrated from nulceus to the soma, also as I perfused the animal 2h after the behavior (so the translocation migh be proceeded).

I will try again and see :)

Thanks again for your help,

Best.

Hi Noemie,

from what I have seen in the basolateral amygdala, c-Fos staining is generally nuclear, but I have seen also more cytoplasmic signal, particularly in some interneuron populations. We interpreted this as biological and not artifactual. However, if you see no nuclear staining at all, either there is no strong neuronal depolarization in the area you are examining prior to perfusion, or something might be wrong with the immunostaining.

We used an anti-c-Fos antibody raised in rabbit (1:500; catalog #ab7963, Abcam) revealed with an anti-rabbit Cy3 secondary (1:500). Sections were 4% PFA fixed, 60um thick and not frozen. Incubation was done in PBS + 0.3% Triton X-100 to improve permeability. Slices were not dehydrated prior to mounting. Perhaps this is something you could try if you have doubts about the current immunostaining.

My other concern is the choice of the DAPI staining as we found c-Fos signal also in glia and not only in neurons. With DAPI you would not be able to discriminate between the two. I would rather quantify c-Fos immunoreactivity in combination with a clear cytoplasmic marker that is either neuron specific or that allows you to discriminate between neurons and other cells based on cytological features.

We used Nissl staining. For example this paper gives useful hints on how to recognise neuronsArticle Distinction of Neurons, Glia and Endothelial Cells in the Ce...

NeuN staining could be another option.

Happy to provide further details if you need.

Cheers,

Marco

Hi Marco ! Thanks for your reply ^^ I discussed today with Prof. Capogna and he also told me that in amygdala it should be nuclear staining.

But he was not surprised too to see cytoplasmic labeling. I am currently trying to reproduce the IF. I might change the concentration of the c-fos (I will try 1:500).

Let´s see ^^

Best !

If this is the antibody you are using https://www.cellsignal.com/products/primary-antibodies/c-fos-9f6-rabbit-mab/2250, the company recommends a pretty drastic dilution (1:6400) for immunohistochemistry. I don't know what you used previously, but not sure 1:500 would work well...

I used the same dilution as this paper published in Nature Comm (1:200 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403372/) and now I am using the dilution (1:500) from this paper (http://www.nature.com/articles/ncomms11096?proof=true) and it seemed to work well for them. I was surprised too to see the high dilution recommanded by the company..

Hi Noemie, it is not easy to find a good working c-fos antibody with a clear nuclear expression. If you have cytoplasmic staining it is hard to quantificate this cells especially if you count them automatically. The antibody, that I prefer and recommand at this moment is a c-fos ab form synaptic systems (www.sysy.com; #226 003; positiv controls: nucleus paraventricularis or supraoptici; If you are working with 30µm or thicker sections I recommand the free floating methode. advantage: your sections get in contact with the antibody from both sides. This leads to a stronger staining result. To find out which delution is the best for your protocol make a delution raw of different delustions. After you have finished your IHC or IF mount the sections on slides, air dry them at 4°C in the dark over night. Now you can mount them with any mounting medium which is suitable for IF. Good luck!

Dear all, Thanks a lot for your answers ! I saw nuclear labeling on some slices too which is a good sign and recently I changed the dilution to 1:500 which is better. I am currently reproducing the data to make a range of dilution and to have negative controls. But taken together, the experiment seems to work :)

And I am happy to know that the dehydration with the toxic Xylen is not an essential step !

Best

I'm encountering a similar issue where I have made multiple attempts to address the problem. Despite changing three different antibodies, I continue to observe consistent staining patterns. Specifically, on the right side, the staining is localized to the nucleus, while on the left side, the staining appears to be distanced from the nucleus.