[Histonet] Subject: wheat germ agglutinin
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
I did not describe routine de-wax, wash in PBS ect.
Novartis Cambridge M6170871-7613.
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