Confocal for AP

From:Gayle Callis

For confocal applications, you will find this listserver invaluable,

Also contact Molecular Probes technical services, they also have a Zeiss
510 META confocal scanning microscope and certainly should be able to help
you with application, or tweaking the system since fluorescence is their

Not sure if available, but antibody to detect AP (anti alkaline phosphatase
-biotinylated, or antiAP, come back with biotinylated secondary and detect
with Strepavidin Alexa 488 or SA 546.)  As for fixation, you can try
several to determine best results.  Acetone, Acetone 75%/absolute ethanol
25% on overnight air dried cells, fix at RT next morning for 5 minutes,
rinse immediately with TBS.  Stay away from PBS as phosphate ions are
contaminates for AP, if left in cells - could cause diffuse nonspecific

For CLSM work here, am contemplating trying Vector Red (Vector
Laboratories), an AP substrate that is red with light microscopy and
fluoresces red at 543 nm excitation.  It is permanent, apparently doesn't
photobleach when excited several times. 

Caveat: to get the best fluorescence with Vector Red - do not develop to
full red color, just a hint of red or pink to the eye and use the
microscope to control the color development. Tween 20 should be added to
buffer mix (VECTOR kit insert tells how to do this) to make fluorescing
chromogen crisp,clean, and less diffuse rather than a heavier red ppt. 

Just make sure you use a good anti fade mounting media properties when
using SA-Alexa fluorochromes for CLSM. 

You wrote: 

I have a colleague interested in staining cytospun cells for Alkaline
phosphatase to localize it in her cells.  Then to analyze it using
confocal microscopy.  Does anyone have any advice on how to fix the
cells and a suitable Ab or kit?  She has tried the ELF 97 kit from
molecular probes with no success.

Good luck and tell us your results

Gayle Callis
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)


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