# Spinal cord staining without fixation

**URL:** <https://forum.painresearcher.net/t/spinal-cord-staining-without-fixation/1101>\
**Category:** Protocols\
**Tags:** spinal\_cord, immunohistochemistry\
**Created:** [June 5, 2023, 1:07am UTC](https://forum.painresearcher.net/t/spinal-cord-staining-without-fixation/1101 "2023-06-05T01:07:53Z")\
**Posts on this page:** 4\
**Page:** 1

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**Author:** ![Zoei](https://avatars.discourse-cdn.com/v4/letter/z/3e96dc/32.png) [@Zoei](https://forum.painresearcher.net/u/Zoei)\
**Post date:** [June 5, 2023, 1:07am UTC](https://forum.painresearcher.net/t/spinal-cord-staining-without-fixation/1101/1 "2023-06-05T01:07:53Z")

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Hello everyone,  
Has anybody immunostained live spinal cord without fixation?

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**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\
**Post date:** [June 5, 2023, 11:49am UTC](https://forum.painresearcher.net/t/spinal-cord-staining-without-fixation/1101/2 "2023-06-05T11:49:51Z")

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A whole cord? A slice? You mean like after a slice recording? I think most people fix. What are you trying to achieve? Why do you want unfixed, live tissue?

Some people use IB4 staining on live cultured DRGs to identify IB4+ neurons @dmolliver

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**Author:** ![Zoei](https://avatars.discourse-cdn.com/v4/letter/z/3e96dc/32.png) [@Zoei](https://forum.painresearcher.net/u/Zoei)\
**Post date:** [June 28, 2023, 8:48pm UTC](https://forum.painresearcher.net/t/spinal-cord-staining-without-fixation/1101/3 "2023-06-28T20:48:25Z")

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That was a misunderstanding from my side. Actually the cord is isolated from formalin-fixed carcasses. Do you have any comment on that?

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**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\
**Post date:** [June 30, 2023, 12:25pm UTC](https://forum.painresearcher.net/t/spinal-cord-staining-without-fixation/1101/4 "2023-06-30T12:25:33Z")

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so you have a formalin fixed spinal cord segment? You need to section this somehow and then do standard immunohistochemistry.

The standard process for most of us is:

1. Fix animal with PFA (not formalin, but they’re roughly equivalent IMO)
2. Cryoprotect with sucrose 30%
3. Make cryosections on a cryostat, either free floating or slide-mounted
4. Standard IHC immunostaining
5. Image