Re: [Histonet] Subject: wheat germ agglutinin

From:Gayle Callis

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:
>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).

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 
>some salts).

>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 
set alone.

  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.

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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