<p>Let's work backwards.</p>

<blockquote>
  <p>I don't currently have access to means to use transgenic orexin knockout mice as are mentioned in the article by Tsujino &amp; Sakurai (though if anyone's used them before I'd be interested to know for the future)</p>
</blockquote>

<p>Just so we're on the same page: orexin knockout mice lack orexin receptors. Have you considered finding a highly selective orexin antagonist? Very loosely speaking, a good antagonist ought to instantiate the functional equivalent of the receptor knockout. </p>

<blockquote>
  <p>Orexin antagonists, especially those that block OX2 or both OX1 and OX2 receptors, clearly promote sleep in animals, and clinical results are encouraging: Several compounds are in Phase III trials. As the orexin system mainly promotes arousal, these new .... Orexin peptide knockout mice also seem to have severe sleepiness, with an inability to maintain long bouts of wakefulness during their active period (50, 51).</p>
</blockquote>

<p>Scammell, T. E., &amp; Winrow, C. J. (2011). Orexin receptors: pharmacology and therapeutic opportunities. Annual review of pharmacology and toxicology, 51, 243-266.</p>

<blockquote>
  <p>So, I'm wondering if anyone is familiar with a substance/approach to determine the approximate amount or volume of intact orexin neurons of the hypothalamus in WT mice (for comparison between animals) using a staining technique.</p>
</blockquote>

<p>Yes, there's a number of methods. One of the common methods is using Fos immunohistochemistry. A summary is as follows:</p>

<p>An immediate-early gene called c-fos will be expressed as a consequence of neuronal excitation. If you administer an orexin agonist, e.g. modafinil (actually perhaps something far more selective than that) or subject the mice to your orexin-activating intervention, then the neurons where that receptor is present ought to upregulate c-fos express when they fire.</p>

<p>You then cut up your rat into fine slicies and apply a high-contrast stain that will bind to the c-fos protein... It's something to do with the antigen-binding site of the protein, but this is all very vague to me without a reference on hand. You then wash the rat slices. If performed correctly, the stain will remain where the c-fos has been expressed.</p>

<p>You then spend many, many hours counting up the stained dots. You compare this against a control, and the difference allows you to reasonably infer where the orexin receptors are.</p>

<p>The following may help:</p>

<p>Estabrooke, I. V., McCarthy, M. T., Ko, E., Chou, T. C., Chemelli, R. M., Yanagisawa, M., ... &amp; Scammell, T. E. (2001). Fos expression in orexin neurons varies with behavioral state. Journal of Neuroscience, 21(5), 1656-1662.</p>

<blockquote>
  <p>I'm part of a large lab studying other conditions that share some symptoms with narcolepsy, and am interested in looking at orexin-producing neurons in our animal models…  So, I'm wondering if anyone is familiar with a substance/approach to determine the approximate </p>
</blockquote>

<p>This is what I don't get: why don't you ask your supervisor and say, "yo, is anyone in our lab doing staining?" -- maybe they are, maybe they aren't. If not, you ask your supervisor, "who among your colleagues does staining?" And they will know or know someone who knows. You then get in touch with said researcher. How it works is that you get taught the staining technique, and in exchange, they get free labour. Alternatively, if they're not down with that arrangement, you simply ask them that the next time they're doing staining, if you can drop by and just watch. It's a simple courtesy that they accommodate your request, and I guarantee you that you will learn far more in one afternoon than you could hope to on SE.</p>

CC BY-SA 3.0
2017-12-30T20:30:11.167Z
2017-12-30T20:30:11.167Z
faustus
<p>I am interested in studying possible depletion of orexin levels in the brains of some mice, and am wondering if anyone knows of a staining substance/technique for use on slices of fixed mouse brains that would highlight orexin-producing neurons of the hypothalamus such that their number/volume could be examined.</p>

<p><strong>Context</strong> - <a href="https://en.wikipedia.org/wiki/Orexin" rel="nofollow noreferrer">Orexin</a> is also known as hypocretin. It is a neuropeptide associated with sleep regulation that's produced primarily in the hypothalamus. It was originally discovered in rats and it's important to me because it is well-known that depleted orexin-producing neurons are connected to narcolepsy (<a href="https://en.wikipedia.org/wiki/Orexin" rel="nofollow noreferrer">Tsujino &amp; Sakurai, 2009</a>). I'm part of a large lab studying other conditions that share some symptoms with narcolepsy, and am interested in looking at orexin-producing neurons in our animal models. I don't currently have access to means to use transgenic orexin knockout mice as are mentioned in the article by Tsujino &amp; Sakurai (though if anyone's used them before I'd be interested to know for the future), but I do have means to stain, view, and photograph fixed brain slices from our existing animals.</p>

<p>So, I'm wondering if anyone is familiar with a substance/approach to determine the approximate amount or volume of intact orexin neurons of the hypothalamus in WT mice (for comparison between animals) using a staining technique.</p>

<p><sub>
Tsujino, N., &amp; Sakurai, T. (2009). <a href="https://en.wikipedia.org/wiki/Orexin" rel="nofollow noreferrer">Orexin/hypocretin: a neuropeptide at the interface of sleep, energy homeostasis, and reward system.</a> Pharmacological reviews, 61(2), 162-176.
</sub></p>

<neuroimaging><animal-cognition><experimental-neuroscience>
How to stain for orexin neurons in mice?
ArtemisPondering
# How to stain for orexin neurons in mice?

(Asked by: username_0 on 2017-08-09)

I am interested in studying possible depletion of orexin levels in the brains of some mice, and am wondering if anyone knows of a staining substance/technique for use on slices of fixed mouse brains that would highlight orexin-producing neurons of the hypothalamus such that their number/volume could be examined.

**Context** - [Orexin](https://en.wikipedia.org/wiki/Orexin) is also known as hypocretin. It is a neuropeptide associated with sleep regulation that's produced primarily in the hypothalamus. It was originally discovered in rats and it's important to me because it is well-known that depleted orexin-producing neurons are connected to narcolepsy ([Tsujino & Sakurai, 2009](https://en.wikipedia.org/wiki/Orexin)). I'm part of a large lab studying other conditions that share some symptoms with narcolepsy, and am interested in looking at orexin-producing neurons in our animal models. I don't currently have access to means to use transgenic orexin knockout mice as are mentioned in the article by Tsujino & Sakurai (though if anyone's used them before I'd be interested to know for the future), but I do have means to stain, view, and photograph fixed brain slices from our existing animals.

So, I'm wondering if anyone is familiar with a substance/approach to determine the approximate amount or volume of intact orexin neurons of the hypothalamus in WT mice (for comparison between animals) using a staining technique.

Tsujino, N., & Sakurai, T. (2009). [Orexin/hypocretin: a neuropeptide at the interface of sleep, energy homeostasis, and reward system.](https://en.wikipedia.org/wiki/Orexin) Pharmacological reviews, 61(2), 162-176.

**Tags:** neuroimaging, animal-cognition, experimental-neuroscience


---

## 1 answers

### Answer by username_1 on 2017-12-30. Score: 1

Let's work backwards.

> I don't currently have access to means to use transgenic orexin knockout mice as are mentioned in the article by Tsujino & Sakurai (though if anyone's used them before I'd be interested to know for the future)

Just so we're on the same page: orexin knockout mice lack orexin receptors. Have you considered finding a highly selective orexin antagonist? Very loosely speaking, a good antagonist ought to instantiate the functional equivalent of the receptor knockout.

> Orexin antagonists, especially those that block OX2 or both OX1 and OX2 receptors, clearly promote sleep in animals, and clinical results are encouraging: Several compounds are in Phase III trials. As the orexin system mainly promotes arousal, these new .... Orexin peptide knockout mice also seem to have severe sleepiness, with an inability to maintain long bouts of wakefulness during their active period (50, 51).

Scammell, T. E., & Winrow, C. J. (2011). Orexin receptors: pharmacology and therapeutic opportunities. Annual review of pharmacology and toxicology, 51, 243-266.

> So, I'm wondering if anyone is familiar with a substance/approach to determine the approximate amount or volume of intact orexin neurons of the hypothalamus in WT mice (for comparison between animals) using a staining technique.

Yes, there's a number of methods. One of the common methods is using Fos immunohistochemistry. A summary is as follows:

An immediate-early gene called c-fos will be expressed as a consequence of neuronal excitation. If you administer an orexin agonist, e.g. modafinil (actually perhaps something far more selective than that) or subject the mice to your orexin-activating intervention, then the neurons where that receptor is present ought to upregulate c-fos express when they fire.

You then cut up your rat into fine slicies and apply a high-contrast stain that will bind to the c-fos protein... It's something to do with the antigen-binding site of the protein, but this is all very vague to me without a reference on hand. You then wash the rat slices. If performed correctly, the stain will remain where the c-fos has been expressed.

You then spend many, many hours counting up the stained dots. You compare this against a control, and the difference allows you to reasonably infer where the orexin receptors are.

The following may help:

Estabrooke, I. V., McCarthy, M. T., Ko, E., Chou, T. C., Chemelli, R. M., Yanagisawa, M., ... & Scammell, T. E. (2001). Fos expression in orexin neurons varies with behavioral state. Journal of Neuroscience, 21(5), 1656-1662.

> I'm part of a large lab studying other conditions that share some symptoms with narcolepsy, and am interested in looking at orexin-producing neurons in our animal models… So, I'm wondering if anyone is familiar with a substance/approach to determine the approximate

This is what I don't get: why don't you ask your supervisor and say, "yo, is anyone in our lab doing staining?" -- maybe they are, maybe they aren't. If not, you ask your supervisor, "who among your colleagues does staining?" And they will know or know someone who knows. You then get in touch with said researcher. How it works is that you get taught the staining technique, and in exchange, they get free labour. Alternatively, if they're not down with that arrangement, you simply ask them that the next time they're doing staining, if you can drop by and just watch. It's a simple courtesy that they accommodate your request, and I guarantee you that you will learn far more in one afternoon than you could hope to on SE.


---

