measuring hypocotyls

STEPHEN A ROLFE S.Rolfe at sheffield.ac.uk
Mon Feb 27 15:24:31 EST 1995


On 25 Feb 1995, STEPHEN A ROLFE wrote:

> Dear netters,
> 
> I have an undergraduate project student in the lab who is examining 
> factors affecting hypocotyl elongation in Arabidopsis.  Does anyone 
> have a good staining protocol so that cells within the hypocotyl can 
> be visualised easily?  The aim is to distinguish between cell 
> expansion and cell division so we wish to be able to measure both 
> cell number and cell length.

(I am trying to look at whole hypocotyls)

Here are the replies I received.  Thank you all very much for your help

Steve Rolfe
//////////////

From: James Masucci <masucci at biology.lsa.umich.edu>

Although I haven't paid much attention to the hypocotyl, we have measured 
cell length and cell number in the root epidermis.  I find that staining 
usually is not necessary.  Two stains that you can try are Toluidine Blue 
and FB28 (fluorescent brightener 28, from sigma).  FB28 stains cell 
walls but you need a fluorescent microscope to use it.  I think some 
people use ruthenium red, but I have no experience with that.  Our 
staining protocols are quite crude.  We add a little bit of stain to 
"artificial pond water" and let seedlings soak until they are done.  I 
can give you some more information if you are interested.

Good luck.

Jim Masucci
masucci at umich.edu
//////////////////
From: "David W. Meinke" <meinke at osuunx.ucc.okstate.edu>

Stephen:

You might try the clearing solution used to examine young seeds.  The 
recipe can be found in: Developmental Biology 165: 566-573; and 
Development 120: 3235-3245, or in my chapter on seed development in the 
new book on Arabidopsis published by Cold Spring Harbor Press.  

Regards,

-----
David W. Meinke
Department of Botany
Oklahoma State University
Phone: 405-744-6549
FAX:   405-744-7673
 
Original Email Address Has Been Changed.
Please Note Updated Email Address Below:
 
meinke at osuunx.ucc.okstate.edu
///////////////////////////////
From: ajt at rri.sari.ac.uk (Tony Travis)

Try 2.5g borax + 2.5g Toluidine blue in 100 ml water.

For more details see Travis, A.J., Murison, S.D.M. and Chesson, A.
(1993)  Estimation of plant cell wall thickness and cell size by image
skeletonization, J. Ag. Sci., 120, 279-287.  You may also be interested
in the image analysis methods we used.

	Tony.
--
Dr. A.J.Travis,                       |  JANET: <ajt at uk.ac.sari.rri>
Rowett Research Institute,            |  other: <ajt at rri.sari.ac.uk>
Greenburn Road, Bucksburn,            |  phone: +44 (0)224 712751
Aberdeen, AB2 9SB. UK.                |    fax: +44 (0)224 716687


--
Dr. A.J.Travis,                       |  JANET: <ajt at uk.ac.sari.rri>
Rowett Research Institute,            |  other: <ajt at rri.sari.ac.uk>
Greenburn Road, Bucksburn,            |  phone: +44 (0)224 712751
Aberdeen, AB2 9SB. UK.                |    fax: +44 (0)224 716687
///////////////////////
From: Matt J B Geisler <mgeisler at magnus.acs.ohio-state.edu>

     This depends, do you want to work with fresh tissue or fixed tissue? For 
fresh tissue, I would suggest either Crystal Violet (1%) or clearing in 
80%ethanol followed by staining in 100% ethanol and 1% safranine (this will 
take 1 hour for weak staining or 1 day for good staining).

     If you want more elaborate staining, try pre-treating in 2% Ferric 
Ammonium sulphate, 3 washes in distilled water, then in 0.5% hematoxylin. All 
of the cellulose should go very dark. You may need to clear the tissue first, I
would reccomend autoclaving in lactic acid.

     For further ideas, Try McCulley and O'Brein's book on light micrography 
and techniques (forget exact title, but I can look it up for you)

					Fellow Arabidopsis microscopist
							    Matt
///////////////////
From: Stuart Baum <sfbaum at ucdavis.edu>

hello steve,
	are you looking at whole hypocotyls? if so, try clearing the 
tissue in lactic acid, or NaOH and then slit the hypocotyl in half and 
look at it unstained under a microscope.  this should work, but if not, 
try using a dilute conc. of toluidine blue (ca. 0.25%) after clearing the 
tissue.
	if you need more help, i'll be happy to help...sincerely
				stuart baum
/////////////////



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