Catherine

We have used TRITC-Con A at 20 microg/Ml in PBS without any problems.
I append the reference and a piece of its M&M below.

Hope this helps

Michal


Reference:
Opas, M., L. Fliegel, E. Dziak and M. Michalak. 1991:  Regulation of
expression and intracellular distribution of calreticulin, a major calcium
binding protein of non-muscle cells. J. Cell. Physiol. 149, 160-171.

Relevant text:
Polyclonal antibodies against calreticulin were used at a 1:50 dilution in
phosphate?buffered saline (PBS), while purified IgGs were used at a
concentration of 200 5g/ml. Higher dilutions of antisera were tested (up to
1:200) but no improvement of background staining was observed under these
conditions except for same decrease in the specific signal. Therefore, the
dilution of 1:50 was used as a standard in this study. FITC?conjugated
secondary antibodies (rabbit anti?sheep and donkey anti?goat) IgGs (used at
1:30 in PBS) were purchased from Bio/Can (Mississauga, Ont.).
TRITC?conjugated Con A (Sigma) was used at 20 µg/ml in PBS. For double
labelling fluorescence microscopy cells were fixed in 3.8% formaldehyde in
PBS for 10 min, extracted with RST X?100 solution (0.1% Triton X?100 in
buffer containing 100 mM PIPES, 1 mM EGTA, 4% (w/v) polyethylene glycol
8000, pH 6.9) for 3 min, washed in PBS for 10 min, and then processed for
double labelling with anti?calreticulin followed by FITC?conjugated
secondary IgGs, then followed by TRITC?Con A. All incubations were carried
out for 30 min at room temperature and were followed by a 10 min wash in
PBS. After the final wash, the slides were mounted in vinol 205S (St.
Lawrence Chemical, Toronto, Ont.), which contained 0.25% 1,
4?diazabicyclo?(2,2,2)?octane (Polysciences) and 0.002% p?phenylenediamine
(Fisher) to prevent photobleaching. For quadruple fluorescence microscopy,
the cells were fixed in 3.8% formaldehyde, stained for 30 sec. with acridine
orange (10 5g/ml in PBS) and photographed. Care was taken to bleach the
stain completely, and then the same cells were re?stained for 30 sec. with
DiOC6 (3 5g/ml in PBS) and re?photographed. The coverslip was then removed
and the cells were washed in PBS and extracted with RST X?100 solution for 3
min. After an additional wash in PBS, the cells were re?stained with
anti?calreticulin/FITC?conjugated secondary IgGs, and TRITC?Con A as
described above and photographed.

-----Original Message-----
From: Confocal Microscopy List
[mailto:[log in to unmask]]On Behalf Of Catherine
Carolan
Sent: Monday, February 21, 2000 7:36 AM
To: [log in to unmask]
Subject: Concanavalin A (con A)


In connection with the recent discussion on ER probes.

We have recently purchased Alexa labelled concanavalin A (con A) for
visualization of ER in fixed samples. I am reluctant to use the recommended
dissolving solution (0.1M sodium bicarbonate, 1mM Mn2+ and 1mM Ca+). Even at
room temperature this readily precipitates out and so I can't imagine it
being suitable for freezing. Are any of the members of the list using this
stain and if so any suggestions on dissolving buffer and concentrations used
would be much appreciated
Thanks

Catherine Carolan
Dept of Physiology
National University of Ireland, Galway
Ireland

Phone 00 353 91 524411 ext 3665
Fax   00 353 91 750544

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