root stain for confocal

Alessia Para alessia.para at sh.se
Tue Sep 25 06:24:13 EST 2001


Hi,
I'm having a lot of problems with the PI staining for root tips to be
visualised by confocal.
The protocol I follow is found in the web page for cell ablation of Utrecht
university (http://www.bio.uu.nl/~mcbroots/laser.htm) but it is not very
detailed so I've tried to manage myself to guess what is not reported.
As they suggest I use a 10microgram solution of PI (should it be dissolved
in water or some other kind of solution like L-arginine pH 12 as I use for
nuclear staining? I have in water and it seems to work) to make the cell
wall evident.
I spot 100 microliters of PI solution on a  coverslip (we use a round frame
to hold a coverslip) , place the seedlings (I used 2, 3,4 day and 1 week
old seedlings ) in it and cover the roots with a square coverslip to let
the cotyledons out .
It used to work at once but now the very same procedure doesn't work any
more (low or no penetration inside the root).
I've tried to change the PI solution but nothing happened.
Do you have any idea about the parameter that can influence the penetration
? Could the pH  (the water I use has a pH around 7) or the temperature
have an effect?
Thank you in advance for any suggestion you will give me.

Alessia

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