replies to confocal protocol query

Philip N. Benfey benfeyp at ACFCLUSTER.NYU.EDU
Thu Jun 17 10:40:28 EST 1993


There were relatively few replies to my request for staining protocols applicable to confocal
microscopy.  This indicates either that this technique is under-utilized in the Arabidopsis
community or that people are jealously guarding their protocols.  I have tried three protocols,
two of which did not come through the net but were from Steve Ruzin at Berkeley.  One was
a vital stain using fluorescein diacetate with which I was able to see a fair amount of cellular
detail but was somewhat variable in the intensity of staining.  The other involved fixation and
used acriflavine.  This allowed me to image quite a bit of cellular detail.  These protocols can
be obtained directly from Steve at ruzin at nature.berkeley.edu.  He was a bit hesitant to allow
this information to go out through the net for fear of being deluged with requests.  So, if you
don't need the protocol immediately, please don't ask for it.  The same goes for the protocol
using Propidium Iodide provided by Mark Running (see below).  He will provide it directly to
anyone interested.  While it appears to be an effective staining protocol, it is rather time
consuming (approximately 2 weeks).

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From: IN%"DGALBRAI at CCIT.ARIZONA.EDU" 13-APR-1993 15:07:10.51


We had a lot of success using R-18 (rhodamine B coupled to a C18 hydro-carbon
tail) for highlighting membranes.  R-18 is usually excited at around 540nm
using the mercury line, but can be excited by the argon line at 514 nm.  
We sysnthesized R18 ourselves, but you can buy it from Molecular Probes.
It works best under conditions that allow its partition into the membranes
over a reasonably long period (i.e. overnight in cell suspension cultures).
I would expect that it could be used with roots or root cultures.  

Good luck

David

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From: IN%"fwu at cabell.vcu.edu"  "Fang-Sheng Wu" 19-APR-1993 10:01:06.74

I have used rhodamine 123 to visualize the mitochondria with
confocal microscope.  Individual mitochondria were clearly visible
in living root tips of Brassica.  To visualize nuclei, I used DAPI.
The procedure is similar to what I have described in Planta
171:346-357(1987).
Fang-Sheng Wu

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From: IN%"RunningM at starbase1.caltech.edu"  "RunningM" 13-APR-1993 01:20:50.79

I have developed a protocol for staining plant nuclei with propidium iodide,
which I will enclose after this note.  The procedure was intended for flowers,
but it should work with any tissue.  The protocol generally works well, but it
is not 100% effective.  Should you try it, I will be interested in any problems
or suggestions you might have.  Feel free to contact me at any time about it. 
We have almost completed a paper that contains the protocol and several
confocal images, and it should be submitted within the next week.  

Mark Running, Meyerowitz laboratory





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