RE: [Histonet] Fluorescent cytoplasmic marker

From:"Monfils, Paul"

How about using a lectin instead of an antibody?  They can be purchased
either directly conjugated or biotinylated, so the same ABC systems, either
peroxidase or fluorescent, that are used with antibodies can also be used
with lectins.  In my lab we do quite a bit of lectin staining, using a
battery of about 35 different lectins.

For those who might not be familiar with lectins, they are proteins or
glycoproteins which bind strongly to specific sugars, more or less analygous
to the way antibodies bind to specific proteins.  Therefore they can be used
to identify and localize sugars in cells and tissue sections, analygous to
identifying and localizing cell proteins with antibodies. Compared with
antibodies they are cheaper to buy, easier to use (no primary and
secondary), less dependent on the type and extent of fixation, and yield
results that are visually similar.  It is also possible to use an
unconjugated lectin followed by a secondary antibody against the lectin, but
in my opinion that's doing it the hard way.

Cytoplasm is typically rich in the sugar mannose, and the lectin Con A which
binds to mannose therefore stains cytoplasm strongly.  This has been
consistent in my lab in many kinds of cells from a variety of species.
Another good lectin for strong general cytoplasmic staining is WGA, which
binds to sialic acid, another common cytoplasmic component.

Paul M.

> ----------
> From: on behalf of
> Pixley, Sarah (pixleysk)
> Sent: 	Tuesday, September 20, 2005 11:56 AM
> To:
> Subject: 	[Histonet] Fluorescent cytoplasmic marker
>  Dear Histonetters:
> I need an antibody (or a stain) that will label all of the cytoplasm of
> every cell. For example, a reliable (and hopefully cheap) antibody
> against some common housekeeping protein that we can then localize with
> a fluorescent secondary antibody. I want to be able to use the confocal
> microscope to visualize the entire cytoplasm of every cell, because we
> are asking questions about where the cells are relative to an unusual
> substrate. We have tried staining for alpha and beta tubulin and the
> filamentous staining is just not sufficient. We want to fill the
> cytoplasm with a fluorescent marker. And we don't want to have to
> transfect the cells or inject them. These are mouse cells.
> Thanks,
> Sarah Pixley
> Univ. Cincinnati
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