Cynthia, 

You should join the confocal listserver CONFOCAL@LISTSERV.BUFFALO.EDU and
ask this question to some confocal experts for specific questions about
confocal laser scanning microscopy.  

There are ways to stain a whole salivary gland and visualize where the
antibody localizes using two color immunofluorescent staining - can even be
done on a live animal. It would not be an easy task and if that is what she
wants to do on a whole thick salivary gland, may not be able to do to
thickness. I have a great publication on how that was done with two photon
confocal. 

Vibratome sections can be IFA stained without fixation and levels for
autofluoresence in tissues must be taken into account although can provide
interesting, nice contrast if one detects the Borrelia with a very bright
red fluorophore (Alexa 546, Molecular Probes). In order to see if the
Borrelia are inside cells, the counterstain should be a nuclear stain that
fluoresces.  DAPI will work unless you do not have the correct laser that
excites DAPI, we can't do that with our 510 META - it requires another
laser.  There is another nulear dye from Molecular Probes, TOPRO3 that
excites in far red, 633 nm, but you can set the color to look like DAPI,
rather than a red (can do some great things with CLSM as long as you say so
in publication!) 
This will light up the nuclei very nicely. Molecular Probes has some
elegant products and their 9th edition handbook is a joy to have in the
lab, interesting reading.   

Good luck on seeing something in that whole salivary gland.  I think she
may have to do frozens or vibratome sections and NOT prefixed with acetone.  


All,

A post doc came by yesterday with a question. I am asking for input. 

She has removed Borellia infected salivary glands from mice, fixed with
acetone and put on slides. She now wants to do Immuno-flourescence using the
confocal [ She has a tagged antibody for the Borellia]  She would like to
visualize the salivary gland to see if the Borellia is within cells or
external. I recall some talk of hematoxylin being fluorescent and I think
acridine orange is as well. I have no idea if this will work but she would
like to try something before she starts all over again with frozen or
possible vibratome sections. On searching the archives I did not find
anything encouraging. John Kiernan had some comments and I will check his
book. 

Anyone out there with suggestions please pass them on.

Please resist suggesting they check before staring a project as I believe
that is useless here!

C

Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317





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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu