Fresh tissue sample/Best method - Need your help!

Use for discussion of various microscopy and imaging techniques, such as phase, fluorescence and confocal microscopy, electron microscopy, and various other imaging techniques.

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Fresh tissue sample/Best method - Need your help!

Postby rcs » Jul 13 2010 6:54 pm

Hi all,

I would like to know the best way to visualize fresh skin tissue, I need a technique that allows me to image YFP and mCherry at the same time. No need for staing, as I am using CD11c-EYFP transgenice mice (DCs express YFP) and the parasite I am interested at express mCherry. I also can process the tissue and work only with the dermis if necessary.

I think the best way to do it is with fresh tissue, as the cells/tissue won't shrink and I won't get artifacts. Is a multiphoton microscope the best option or I could use a confocal instead? Would you please give me some examples of scopes I could use?

Thank you VERY much for any help, even if it is a reference or something like that :D
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Re: Fresh tissue sample/Best method - Need your help!

Postby rcs » Jul 14 2010 11:42 am

Forgot to mention, the parasite is fairly large: around 1um x 15um. Thanks! :D
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Re: Fresh tissue sample/Best method - Need your help!

Postby CrowSan » Jul 14 2010 2:24 pm

Sounds like the best way would be to obtain what is called a "skin window". I haven't done it but it uses a device (a bit like a potatoe pealer) that shaves off a thin layer of dermis. I saw it used in haematology quite a while ago. Depending on what resolution you need I would have thought a confocal would be fine.
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Re: Fresh tissue sample/Best method - Need your help!

Postby rcs » Jul 15 2010 2:09 pm

Thanks CrowSan,

That makes sense, I will look into it!

r
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Re: Fresh tissue sample/Best method - Need your help!

Postby CrowSan » Jul 15 2010 3:24 pm

No worries - I think the technique was originally done to remove skin to see the underlying dermis but when I was working in haematology we had an old guy there who used to use it on himself to generate dermal slices he could put under the microscope (we called him "mad dog" for obvious reasons) so I know the skin can be removed and analysed in this way.
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