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 [[log in to unmask]] On Behalf Of Donaldson, Lloyd (ensis, Rotorua)
Sent: Thursday, 30 October 2008 6:09 a.m.
To: [log in to unmask]
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 [log in to unmask]
>>
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
[log in to unmask]
http://core.igb.uiuc.edu
<http://core.igb.uiuc.edu/>
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