Re: [Histonet] Subject: wheat germ agglutinin
Lectins are notorious for binding to more than one tissue component
containing the carbohydrate residue the lectin binds to. Be sure to look
up what WGA binds to in order to know if you are going to have more than
one structure, other than the cardiomyocytes being stained.
At 12:09 PM 5/8/2006, you wrote:
> 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).
To try fluorescent staining, use the WGA-biotinylated, and come back with
Molecular Probes Strepavidin- Alexa 488 or 555. but use avidin/biotin
blocking to quench any endogenous biotin present.
A correct negative control is the inhibiting/eluting sugar
500mMNacetylglucosamine, chitin hydrolysate from Vector, or 100 mM acetic
acid. The sugar is what we use with lectins, and you take your working
concentration of WGA, and dilute it in the 500 mM
N acetylglucosamine, let it incubate for 30 min to 1 hour (we let it
incubate overnight at 4C to ensure a lectin is totally bound to the sugar
and not available to bind to the carbohydrate residue in the
cardiomyocytes. This is added to a section as a negative control.
>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
Do a dilution panel, starting at 40 ug/ml then 20 ug/ml, then 10 ug/ml, 5
ug/ml, etc, etc. 30 minutes should work for incubation.
>What I should use for blocking- sugar or serum or both?
Never use serum with lectins, there are carbohydrate residues in serums
that will bind to the lectin. There is no reason to block with lectins,
they are NOT antibodies. However if you work with biotinylated lectins,
Strepavidin or an ABC system, you should do an avidin/biotin block to
quench endogenous biotin present.
>What I should use as a diluent?
The rinse buffer recommended by Vector is sufficient.
>Vector recommends 1% BSA/PBS, book "Lectin histochemistry" recommended by
>Gayle , as well as J.Kiernan recommend to use lectin buffer (Tris with
>What John referred to IS the lectin buffer which is:
10X Lectin Buffer
Tris 60.57 g
NaCl 87 g
2.03 g Magnesium chloride
1.11 g calcium chloride
in 1 liter distilled water, adjust pH to 7.6 with Concentrated HCl.
Working buffer is either bring this to final volume of 10 L with distilled
or dilute the 10X buffer 1:10 to make working Lectin buffer.
This buffer is simply a routine TBS buffer with calcium chloride and
magnesium chloride added.
If Vector recommends 1% BSA/PBS then use it, but remember that some lectins
will not work well with phosphate ions present, hence the lectin buffer
recommendation. WGA and UEA1 are NOT affected by phosphate ions, so what
Vector recommends should work well.
>May be I should use other fluorescent label?
WGA conjugated to FITC should work well, we had UEA1-FITC direct staining
on M cells in small intestine frozen sections. The section can be mounted
with VECTASHIELD hardset containing DAPI, but for imaging, VECTASHIELD hard
I will appreciate any advices.
> Protocol for biotynilated WGA which I used:
> Block of peroxidase
Yes with a peroxidase system
> Block with 2% BSA/PBS
You are not performing an antibody stain, a protein block is not needed for
> 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.
If you have poor staining, weak, etc - a trypsin enzyme digestion for 10
min at 37C will work on FFPE tissues.
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-4303 (FAX)
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