Fos staining in the spinal cord

#1

Hi everyone,

Looking to see if anyone has had success with fos staining in the spinal cord. I have an antibody that I like, and a protocol that seems to be working okay, but would like to have some improvements. Really trying to do a battery of tests (thermal, chemical, and mechanical assays). Any insight on people’s protocols would be great.

Currently I am anaesthetizing animals and pinching the paw for 30 seconds, and then perfusing 2 hours later. Also want to try this with cold, heat and chemical.

#2

Standard IHC staining. Nothing special. This is the Santa Cruz Fos antibody which isn’t made anymore. 90 mins to 2 hrs is typical time.

Do you have an antibody?

#3

I also used anti-fos from santa cruz before for sc staining after capsaicin injection to hind paw and it worked well, background was clear. The protocol I used was a regular one of IF.

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#4

The cata # is 271243 the one I used.

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#5

I don’t have an antibody - I have one that someone was using in rat tissue, but it’s very background-y in mouse tissue…So I’m kind of back at square one. Any others besides Santa Cruz?

#6

@whshhaibing123 was this in mouse tissue?

#7

Yes, it is the mouse.

#8

@ShanTan
SC made a new one apparently, and someone tested it:
https://www.scbt.com/scbt/product/c-fos-antibody-e-8

There seems to be a need for a good alternative to the original sc-52. Most people are still using stockpiles of that.

Are you committed to IHC or will you consider RNAscope?

Here is some Fos RNAscope I did in mouse SC after capsaicin. It’s awesome.

#9

@achamess Just ordered the new SC one. Fingers crossed. Still in the midst of optimizing my RNA scope probes in my mouse spinal cord tissue, but will definitely keep this in mind. I am trying to colocalize c-fos staining with an extracellular marker that is sensitive to tissue treatments (even Triton ruins the signal) so I am weary to go to RNAscope in replace of traditional IHC for colocalization studies at this point, but I am not going to rule it out. Thank you! Will keep everyone updated and will post pics once the antibody comes in.

#10

@ShanTan Great! Fos staining is such a common thing, I’m sure there will be answer for you. One other thing - I’m sure there are labs at your school that have sc-52. Maybe ask if you can take some if you’re only going to do a few experiments?

Sounds like RNAscope might be a no-go. But if you’re doing just straight RNAscope, w/o IHC, the conditions are worked out. See my past posts for pre-treat. We get great results every time, and so do others. But not usually with IHC afterwards. You may try doing IHC before RNAscope. That’s a possibility, although the stability of the RNA may be somewhat compromised. Just gotta try.