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I'm going to be running a study where we hope to manipulate astrocytic activity in the hippocampus using activator and inhibitor DREADDs, after other manipulations. For our purposes, we will harvest tissue 3 days after our experimental stimulation and will continuously activate the DREADDs (activator or inhibitor) during that period. I'm looking for makers that I can use to assess depressed astrocytic activity as well as augmented activity. FOS will likely have gone back to baseline after 3 days of stimulation. GFAP would catch the augmented activity, but probably not depressed activity? Am I correct? What other markers should I be looking at with which I can quantify and hopefully show some pretty pictures for astrocytic activity to show both increased and decreased activity? Thanks ahead of time.

I purchased a c-FOS antibody from Santa Cruz to test it on the rat brain sections. My rats were perfused in 4% PFA, post-fixed overnight in 4% PFA, sucrose infiltrated for 2-3 days for cryosection.

I decided to use this antibody, sc-52, since it was the next popular one after Millipore's according to Journal of Comparative Neurology's antibody database. Since Millipore's one is discontinued for now, I decided to use sc-52 but didn't turn out to be well.

I tested it with and without antigen retrievals. There are some nucleic signals but very a few. Also signal to background ratio was very low. I visualized by using Alexa-488 anti-rabbit antibody. Interestingly, there are lots of c-fos reactivity in the cells along subventricular zones and choroid plexus epithelial cells only if I didn't do antigen retrieval. This signal was absent in antigen retrieval treated groups. I don't know if the signals are real since compared to the strong signals in the lateral ventricle area, other area was pretty much absent and if there are any, their signals are weaker than those in the lateral ventricle area.

If any of you are experts on using c-fos antibody, could you share your opinion on this? Thanks.

I am looking for a way to visualize activated GABAergic neurons after administration of a drug. I am in search of a promising protocol.

Since the Santa Cruz rabbit anti-cfos antibody (sc 52) has been discontinued, is there anyone using succesfully a different antibody against c-fos in idisco?

Hi, I'm recently trying to validate c-fos antibody staining with optogenetics stimulation.

The wild type mice was injected Syn-ChrimsonR-tdtomato virus with cranial surgery.

(somato-sensory cortex, 5mm diameter)

After curing period (3-5weeks) I checked the virus expression by 2P microscopy.

Almost mice were confirmed that they expressed tdTomato and I gave the stimulus light in to the mice cranial window site.

When I gave the stimulus light, the mice were awake and I put them into the isoflurane chamber and delivery the stimulus light.

(674nm laser was used. 50mW at the end of tip in continuous wave, 10Hz stimulation duty-cycle 10%, 1min stimulus and 1 min rest...X5)

After stimulation, mice were placed into the cage and rest at least 75min (Waiting c-fos expression time).

After rest time, the mice were operated perfusion.

The next,

1- 1day PFA fixation

2 - 100um slice by vibratome

3 - 2h Normal goat serum blocking solution embedding

4 - Primary antibody solution embedding ( Merk anti c-fos Rb) (3day)

5 - Washing by PBST 30min/3th

5 - Secondary antibody solution embedding (Alexa 488 - anti Rb)

6 - Washing by PBST 30min/3th.

But all the IHC imaging, the c-fos was formed filament structure in L1-5 in gray mater and nearly existed in soma.

Furthermore, the c-fos signal was very well distributed in whole brain region (gray mater, white mater...wherever!!)

The distribution phenomenon was same If I added another antibody protein (NF, anti-tdtomato,..)

As I know, the c-fos could not escape the nucleus. Why the c-fos signal was existed in every where? ( I never see the filament like c-fos signal)

+1) I checked the cross talk(bleed through) of fluorescence and there was little signal cross talk that could be ignored.

+2) The mice was not trained for the light stimulation . - I think this could be an clue for this problem but I have no confidence.

In my opinion, even they had a surgery event, grabbing would affect mice physiology (noble experience).

i.e : dread, fear, stress -> it could be classified as emotional stimulation and run or resist would be induce motor and somatosensory stimulation.

I also attached the image for that. (blue : NF, Red : c-fos)

Thanks,

Can anyone suggest a good antibody for c-fos staining of brain tissues (neocortex) preferably species except Rabbit.

Thank you very much in advance

Hi everyone,

We are going to perform some immunohistochemistry using c-fos antibody to look at the expression patterns of fos protein in different brain regions. I know that most people postfixate their brains in PFA for 24 h but I read papers that saying brains should not be kept in postfixation more than 1 hour for c-fos staining and also half-strong fixative should be use for that purpose.

So how long should I kept my brains in post-fixation and how strong should it be?

Hi all,

I want to know how long the fluorescent signals of Fos- or Arc-TRAPed neurons lasts.

TRAP (Targeted Recombination in Active Populations) reference papers shows that the animals were sacrificed one week after injecting tamoxifen or 4-hydroxytamoxifen and behavioral induction. However, I think it is permanent and the fluorescent signals of Fos- or Arc-TRAPed neurons wll be visible after a week. Is it fine to detect FosTRAPed neurons 3 or 4 weeks after behavioral induction?

I am looking forward to hearing from you.

Thanks,

Tae-Yong

Hi everyone,

We will look at c-fos expression in different brain regions. As most of you know any new procedure or administration of a drug induce the expression of c-fos and various anesthetics have different effects. So I am trying to find the best drug combination that will not effect our results. Does anyone have any suggestions for anesthetics that can be safely use in a c-fos study?

I am using the Fos2Trap mice crossed with a Ai14 to see dTomato in fos cells activated by 4OH tamoxifen. The dTomation expression is nice on untreated slices but as soon as I perform an immunostaining against Fos to see the cells activated by retrieval, I don't see the dTomato anymore. Does anyone has some experience with fos immunostaining on the line ? I used a classical fos immunostaining with anti-fos antibody from cell signaling and goat anti rabbit A488. Thank you for your help !