Sectioning Hard Grass Stems
Locked
|
Shiv Mayandi Sivaguru, PhD, PhD |
 
|
Rosemary.White |
Re: Sectioning Hard Grass Stems
|
|
Hi Shiv,
Depending on what you need to do, you could try a sledge microtome, we use this for small tree cross-sections, big tobacco stems and also wheat stems, fresh and dry. You do need a really good, well-sharpened microtome knife, the disposable ones aren't up to it - ditto if you try cryosectioning. For this, we just stick the stems in tissuetek or similar and section them very cold - at least -30 C. The other option is to autoclave in a softening agent, one group here autoclaves rice leaves in KOH before staining rust pathogens with fluorescent wheat germ agglutinin, the older rice leaves are so hydrophobic it's the only way to stain haustoria inside the mesophyll. They are a bit soft after this, but perhaps just autoclaving in water or buffer would help. Maybe the pressure cooker isn't going quite far enough..... cheers, Rosemary Rosemary White CSIRO Plant Industry GPO Box 1600 Canberra, ACT 2601 Australia ph 61 2 6246 5475 fx 61 2 6246 5334 ________________________________________ From: Confocal Microscopy List [[hidden email]] On Behalf Of Donaldson, Lloyd (ensis, Rotorua) Sent: Thursday, 30 October 2008 6:09 a.m. To: [hidden email] Subject: Sectioning Hard Grass Stems Shiv We have successfully embedded wheat straw in Spurr resin, LR White may be better although often it doesn't bind to hydrophobic surfaces so might be a problem. For really dense material like Miscanthus we have never had any success. I suggest two approaches: 1/ If you can collect fresh stems they should be much easier to section (without embedding) than dried material which has become permanently hornified. 2/ If you have to work with dried material then try storing in formalin aceto alcohol for long periods (months). For some materials this results in some softening but it does take a long time (6-12 months). This will also work better if you start with fresh rather than dried material. If you do manage to embed the stem it will likely be silicified so you will go through a lot of glass knives ! Regards Dr Lloyd Donaldson - Senior Scientist SCION - Next generation biomaterials Te Papa Tipu Innovation Park, 49 Sala Street Private Bag 3020, ROTORUA, New Zealand DDI 64 7 343 5581 Fax 64 7 343 5507 email [hidden email] >> Hi all, I have never cut hard woody grass stems (similar strength as small dried bamboo stem). One of our client wish to section and stain them and I have tried couple of ways soak and cryo embedding, cook (pressure cook) and mounting in paraffin (based on a book chapter), but it seems not working well. I am about to try embedding in hard polymer such as LR white or Lowacryl, is there any other method proven to be useful than these approaches. Sorry this is not a confocal question. Thanking you in advance Shiv Mayandi Sivaguru, PhD, PhD Microscopy Facility Manager 8, Institute for Genomic Biology University of Illinois at Urbana-Champaign 1206 West Gregory Dr. Urbana, IL 61801 USA Office: 217.333.1214 Fax: 217.244.2496 [hidden email] http://core.igb.uiuc.edu <http://core.igb.uiuc.edu/> |
|
|
In reply to this post by Lloyd Donaldson
Hiii
Why dont you try OCT (resin) and perform cryotome without fixing the stems .. I did that some time back with Tea Stems and it works fine for me ...But it was more like a freeze fracture ..
On Thu, Oct 30, 2008 at 12:39 AM, <[hidden email]> wrote:
|
| Free forum by Nabble | Edit this page |