Re: [Histonet] Subject: wheat germ agglutinin

From:"John A. Kiernan"

Serum albumin is a glycoprotein, so it doesn't
make good sense to include it in a solution of a
lectin. Go with Brooks, Leathen & Schumacher's
"Lectin Histochemistry", which is an authoritative
source and gives very detailed, easily followed
instructions. As you have noted, they recommend
(p.159) a TRIS buffer with a little Ca and Mg
chlorides added. (For some lectins it's Mn rather
than Mg.) The only thing that's likely to need
adjustment is the concentration of the labelled
lectin. When I worked with fluorescently labelled
lectins I preferred a rhodamine to a fluorescein
label because autofluorescence was less of an
annoyance. You can, however, suppress all
autofluorescence before staining. See Biotech.
Histochem. 77(4): 232 (2002).
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
Galina Deyneko wrote:
> Dear colleagues,
>    I would like to ask some advice regarding WGA.
>    We would like to use WGA for label cardiomyocytes membranes on FFPE mouse hearts for image analysis.I clearly remember a message from Dr. J.Kiernan and agree with his opinion, but my lab head would like to try. I stained cardiomyocytes with biotynilated WGA from Vector with ABC kit and received strong signal from endothelial cells and weak signal on cell membrane (I placed picture on histonet web). Now my lab head would like to try with FITC WGA (Vector). Please, share with me protocols, any prompts will appreciated. In archive I found a lot of questions, but no answers. What dilution or final concentration do you recommend? What I should use for blocking- sugar or serum or both? What I should use as a diluent? vector recommends 1% BSA/PBS, book "Lectin histochemistry" recommended by Gayle , as well as J.Kiernan recommend to use lectin buffer (Tris with some salts). May be I should use other fluorescent label?
>   I will appreciate any advices.
>   Protocol for biotynilated WGA which I used:
>   Block of peroxidase
>   Block with 2% BSA/PBS
>   WGA dilution 1:250 (20 ug/ml final concentration) with 2%BSA/PBS
>   Incubation 1 hour at RT in humid chamber
>   Developing with ABC kit (Vector) for 30 min and DAB for 5 min
>   No counterstaining.
>   I did not describe routine de-wax, wash in PBS ect.
>   Thank you.
>   galina deyneko
>   Novartis Cambridge M6170871-7613.
> ---------------------------------
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