Posted by
Dobeck, Justine on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Embedding-Arabidopsis-Thaliana-for-confocal-tp5954747p5958603.html
Have successfully done confocal using 6 micron paraffin sections of decalcified mouse molar and incisor teeth which had been fixed in 10% buffered formalin. Had used some of the sections to do IHC using DAB as substrate with Vector ABC kit. The only difference in protocols was that quenched in 2% sodium borohydride in PBS for 1.5 hours changing the solution at 45 minutes and then rinsed 5 times in PBS before proceeding with blocking step. Sodium borohydride quenches much of the endogenous autofluorescence from aldehydes. Antibodies were to e and n cadherins and used Alexafluors 488 and 568. You might want to increase concentration of primary and secondary from what used for light microscopy.
Justine
Justine M. Dobeck
Biostructure Core Facility
Rm. 5123
The Forsyth Institute
245 First Street
Cambridge, MA 02142
(617)892-8316
Email:
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From: Confocal Microscopy List [mailto:
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Sent: Monday, January 24, 2011 5:27 AM
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Subject: Embedding Arabidopsis Thaliana for confocal
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Hi!
I´m a student doing my exam-project where we are using Arabidopsis Thaliana
to express a HIV protein (p24) in order to creat an edible vaccine.
Previously I have fixated and embedded (paraffin wax) the tissues to
vizualise it in Light microscope (via IHC). Now I need to do the same
procedure but try to analyze the tissue using confocal microscope. For that
I need to know if I can use the same fixation solution for confocal as I did
for Light microscope which was: egual volume of 0.2M Sodiumphosphate buffer
pH 7.2 and Paraformaldehyde (8%) solution?
And also what kind of embedding should I use for the confocal?
Since I have a lot of material left from my previous work I´m hoping to be
able to use it as it is for the confocal.
Is this possible?
Thank you for replying!
/Anna
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