DIY Histology - Embedding Mounting Block recipes

[Tim @ Backyard Brains]

As part of some ongoing nerve conduction velocity experiments, I have been dissecting the nerves out of earthworms and trying to stain the fibers to get accurate measurements of the nerve diameter sizes. I have tried Golgi stains and cresyl violet, but the dyes are not penetrating the whole tissue very well. I think I may have to mount the nerve in an embedding block and then make small slices.

Making a DIY microtome is fairly simple, but I need to embed the tissue first to prepare for slicing. Any ideas for embedding recipes?

Tim

[Cory Tobin]

> Making a DIY microtome is fairly simple, but I need to embed the tissue
> first to prepare for slicing. Any ideas for embedding recipes?

A couple of questions:

1) The nerve that you want to stain, is it still in the worm? Or you
dissected it out and want to embed just the nerve by itself?
2) Do you want to fix, embed, section, clear, and stain the
tissue/cell? Or just embed, section and stain?
2.1) If you want to fix and clear the tissue, do you have access to a
fume hood and vacuum chamber?

-cory

[Tim @ Backyard Brains]

Yes, I am removing the nerve out of the worm, and just want to embed, section, and stain. I am thinking using paraffin wax might be a good embedding compound.

I am just using ethanol as a preservation, not formaldehyde or other nasty chemicals. In place of the xylene step (which removes fats I believe), I used acetone. Haven't determined whether it's a good substitute yet.

[Nathan McCorkle]

On Thu, Aug 8, 2013 at 9:49 AM, Tim @ Backyard Brains
<t...@backyardbrains.com> wrote:
> In place of the xylene step (which removes fats I believe), I

Is that just because you don't have xylene locally available? Toluene
might be a better substitute.

[Mega [Andreas Sturm]]

I am no expert in staining things, but try adding some chloroform. I heard that stuff heavily penetrates cell walls.

And, don't forget to wear latex gloves, of course. and change them often.

[Jonathan Street]

Latex gloves can cope with ethanol and acetone but should not be used with xylene, toluene or chloroform. I would at minimum use nitrile gloves and would be tempted to double glove for chloroform and toluene.

On 8 August 2013 13:02, Mega [Andreas Sturm] <masters...@gmail.com> wrote:

I am no expert in staining things, but try adding some chloroform. I heard that stuff heavily penetrates cell walls.

And, don't forget to wear latex gloves, of course. and change them often.

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[Cory Tobin]

Paraffin works well for embedding, although I've never used it without
fixing prior to embedding so I make no guarantees.

For clearing I would recommend using Histo-Clear
https://www.nationaldiagnostics.com/histology/product/histo-clear
Works well with paraffin and is non-toxic. You don't need a fume hood
and it actually smells quite nice.

Also, if you want to fix the sample but are hesitant to work with
formaldehyde, there are some non/less-toxic alternatives
http://www.ncbi.nlm.nih.gov/pubmed/22947094

-cory

[Mega [Andreas Sturm]]

Yeah, should have been nitril, NOT latex. Sry