Author Topic: TEM staining  (Read 6436 times)

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Offline bertie1

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TEM staining
« on: December 01, 2005, 03:55:32 PM »
Hi guys,

I have a quick question concerning the staining of grids for the TEM. I'm currently looking at scar tissue, fibroblasts, capillaries and leucocytes and I'm not getting good staining intensity, there is very little differential between the formar background (I'm using slotted grids) and the tissue. Now the UA and PbCitrate I've been using are a little old (+ 6 months give or take) but I was wondering if this may be a feature of this type of tissue especially when using formar. Are there any tips or tricks that might help to increase electron density?

Thanks

Bertie

TEM staining
« on: December 01, 2005, 03:55:32 PM »

Offline excalibur

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TEM staining
« Reply #1 on: December 01, 2005, 04:35:32 PM »
Did you fix with gluteraldehyde, then post-fix with osmium?

I used to embed in araldite, what medium are you using?

Your stains are not old at just six months, if you are referring to the powders. If in solution, try making them up fresh and keep in the fridge no longer than a month or two.
Paula K. Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
8901 S Santa Fe, Suite G
Oklahoma City, OK 73139
405-759-3959
www.excaliburpathology.com

Offline bertie1

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TEM staining
« Reply #2 on: December 01, 2005, 05:21:20 PM »
Hi Paula,

I fix with 4% Para with 0.1% Glute, 2.5% Sucrose in 0.1M PB. We do perfusions and postfix for about 3 hours. After that I secondary fix in OsO4 for 1 hour and then embed in Agar-100 resin (Epoxy resin).

The stains are in solution and I stain in UA for 20 min followed by 10 min in PBcitrate. Is this OK? I know that staining times are variable depending on the solution strength (I use the standard strengths) but I'm wondering if I need to lengthen one of the stain times to increase the contrast. I'm a little unsure if this is done with the UA or the PbCitrate.

Thanks for the prompt reply,

Bertie

Offline excalibur

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TEM staining
« Reply #3 on: December 01, 2005, 08:20:28 PM »
Well, your procedure sounds fine. Just make sure your stains are fresh and maybe increase the times a little. Also try a 2% PFA/2% glut in a different buffer, such as cacodylate.

Remember also you are working with scar tissue which is alot of collagen, with fibroblasts few and far between. Did you do thick sections (1 micron) stained with Toluidine Blue to see what is in your block and you are where you want to be? Since these are tiny blocks, you may want to embed several, do thick sections, and then thin section and stain the ones containing the components you need.

Good luck! :wink:
Paula K. Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
8901 S Santa Fe, Suite G
Oklahoma City, OK 73139
405-759-3959
www.excaliburpathology.com

TEM staining
« Reply #3 on: December 01, 2005, 08:20:28 PM »