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Summary of Sectioning Responses

Meisel, Lee A meisel at OCELOT.RUTGERS.EDU
Fri Sep 16 13:07:51 EST 1994


Dear Netters;

Here is a summary of some of the responses I received about sectioning
Arabidopsis.  I would like to thank everyone who responded to my
questions.   If anyone still has more tips I'd be happy to hear them and post
them.  Happy Sectioning.

Lee Meisel

********************************************************
Hi Lee,
I have just finished sectioning a lot of embryos, where the problems are
a little different than leaves, roots, etc.  I'm afraid I can't answer most
of your questions, but here's what works for us:  I have used Spurr's
resin for a few reasons.  a)it uses a chemical catalyst so in our lab it is
easy to get good polymerization (compared to JB-4 and LR White resins
which use heat or UV to initiate polymerization) and b)if you want thin
sections plastic is better than paraffin.  Using Spurr's, we usually cut
about 1 um sections for light microscopy.  I have been serially
sectioning embryo and have gone all the way through with no trouble. I
use glass knives that end up getting changed frequently (about 2 or 3
knives per sample).  Spurr's is not good if you want to do TEM.   
I fix with GAP (glutaraldehyde:acrolein:paraformaldehyde), followed by
an osmium tetroxide post-fix.  The best fix for you will depend in part 
on the tissue and conditions you want to use.

A really good book (although I don't know how available it is) is "The
Study of Plant Structure, Principles and Selected Methods" by
T.P.O'Brien and M.E.McCully, 1981, Termarcarphi Pty. Ltd., Melbourne,
Australia.

I've been staining with methylene blue (0.5% in 0.5% borate).  Toluidine
blue, pH 11, is good for maize leaves, but bad for Arabidopsis shoot
meristems (the tissue takes up the stain too well, giving really dark
cells without good contrast with the cell walls).

I use cheap, precleaned slides that we coat with gelatin (a lab trick
that I don't know the history of): 
Make up a gelatin solution as follows--

        to 100mls hot water add:
                0.5 gms  Gelatin (let dissolve completely before adding
                                            next ingredient)
                0.05 gms Chromium Potassium Sulfate


Rinse slides in water briefly, blot.. then dip into gelatin solution for
approx.  30 sec., blot on paper towel.  Place in a slide box to dry
overnight.

Good luck, Laura.
********************************************************
Good books:  1. Get Johansen
             2. Read it.
I know this dates me.  This is talking about optical microscope
histology.  Always worked for me.
Good luck.
David Wheat
dwheat at mcimail.com
*******************************************************
Some group readers asked for more detail on sectioning plant tissues. 
My previous article referred to Johansen's ''Botanical Microtechnique''
which was the standard when I was doing this type of work in the 60s 
and 70s.  The best way to find out what is current might be to contact 
someone in a local department of botany.  I presume they still do plant
histology at the optical level.  Some of the solvents we used to use are 
probably no-nos now (we used a lot of toluene).  It's easy to fix, embed, 
and section this type of material.  But the easiest way to learn is to
have someone who is already set up to do it walk you through it.  No
point in getting all the equipment and reagents just for a few sections. 
Fix your material in Formalin/Acetic Acid/Alcohol (FAA).  Vacuum 
infiltrate if its hairy, thick, woody, etc.  Then call a local plant
anatomist and visit her lab for a demo.
Good luck.
David Wheat
dwheat at mcimail.com
*******************************************************
hi lee,
	here are some answers to your numbered questions:

1. glutaraldehyde, yes, fixatives make a differece
2.  historesin
3.  trial and error
4.  this is tough, but experience will help.
5.  look up in mccully and o'brien
6.  i use gelatin coated slides
7.  i use glass knives with no problems
8.  for arab. plastic is a must for good tissue integrity
10. see #5
*******************************************************
For a simple procedure, refer to Lang, Ray, and Ray in Genetics 137:
1101-1110 (1994) (August). If you want a thorough manual, look up
O'Brien and McCully (1981) "The Study of Plant Structure: Principles and
Methods", Published by Termarcarphi Pty Ltd., Melbourne.

--
Animesh Ray
Dept of Biology
University of Rochester
Rochester, NY 14627
ray at ar.biology.rochester.edu
anmr1 at bphvax.biophysics.rochester.edu
Phone:(716)275-8986
FAX:(716)275-2070
********************************************************
Dear Dr Meisel,

I just saw your message with questions on sectioning of Arabidopsis. 
We've had quite some problems, because we started with material
embedded in paraffin. This is O.K. for mature tissues, but for young
growing seedlings paraffin or paraplast cannot be used, since these
embedding media cause poor tissue preservation. We therefore prefer to
use plastic embedding media. Depending on the application
(immunocytochemistry, electron microscopy, in situ hybridization....)
we use diferent resins. For normal light microscopy we use Historesin
(Leica, formely Reichert/Jung I believe) which is cut dry (3-10
microns) on glass knives on an ultramicrotome, or any high quality
microtome suitable for glass or diamond knives. Historesin is quite 
hydrophyllic, so resin blocks have to be kept under vacuum with
silicagel. For more general applications (EM, light microscopy,
immunocytochemistry on sections) we also use LR white 
or Lowycryl. These resins are easier to section (although they are a bit
more toxic than Historesin). Finally for in situ hybridization and
immunocytochemistry we also use Butyl/methyl-methacrylate (for
publication see: Kronenberger et al. 1993: Cell Biol. Int. 17
(1013-1021).
As for the fixation, if you just need good tissue preservation use 
glutaraldehyde (1%, EM grade) if necessary in combination with
formaldehyde (4% FA, prepared freshly from paraformaldehyde, don't
use a buffer containing Cl ions in combination with formaldehyde). For 
immunocytochemistry we usually avoid glutaraldehyde.
I'm afraid that histocytochemistry techniques usually take a lot of
time. I can only advise you to go to a lab where these techniques are
already used.

In the meantime I wish you good luck.

Jan Traas
INRA Laboratoire de Biologie Cellulaire
Route de Saint Cyr
78280 Versailles cedex
France
********************************************************





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