Plant Root Sectioning - Microtome sections of roots embedded in parafin (Feb/01/2008 )
I am interested in getting thin sections of roots for a study of the root tissue structure of my plants. In the literature hardening resins like HistoResin/Technovit are widely used to enable section of widths 3-5μm. In my case I think even sections of approximatelly 10μm would be ok. If I will try to embed my dehydrated samples in paraffin instead, inorder to have a more cost effective project, how thin sections will I be able to get with a sledge microtome? Will there be any other disadvantages from the use of parafinn - currently I am preserving my root samples in standard 4% FAA?
We are using the following protocol to fix root material:
Fix roots in AFA (aqueous ethanol (70%): formol (40% aqueous formaldehyde): glacial acetic acid = 90:5:5); usually overnight
When fixed, dehydrated in an ascending ethanol series ( 30%, 50%, 70%, 90%, 95% then 100% and again 100% min. 10 - 15 min each)
embed in paraffin
we are using a cryo-microtome and get very good result with this method. But maybe freezing your paraffin-block prior to cutting it may help...some people at my lab have done this when the "cryo" part of the microtome was not working; they said that they got acceptable results with this method too...
I know that parafin has been the standard means of getting thin sections for quite a long time (and I think I have a good set of protocols for dehydration and embedling). What worries me is how thin the sections can be - definetely thicker than with resins. I would like to get sections around 5um? Could it be possible?
I am afraid I don't have acces to a cryo-microtome. I think I will have to use a sledge one.
i remember using a sledge for large scale brain work - whats wrong with a normal microtome?
btw- cold can crack the wax = less quality
the cryostat is built for a completely different different method of embedding (oct-non fixed- keep in -80)
i think you are over complicating a rather basic problem
cutting at 5um (frozen or wax) takes a lot of skill - i would recomen 7um
7um is fine. I had never planed to use cryotomy anyway. Thank you Dominic.