You wrote:  

Hi All
I dong some lectin IHC and was curious as to what people are using.  want
to use ulex to label brain vasculature and determine micro-vessel density.
Would you recommend the "biotinylated ulex" or an "anti-ulex antibody"  The
literature is not so clear ( writers just don't put in a lot of effort into
writing  IHC material & methods)
 
Thanks In advance 
Luis

If you use an antiUEA antibody, you must apply UEA to the section first.
Lectins are naturally occuring proteins (from plant source, some are
extremely toxic) and glycoproteins the seclectively bind to carbohydrate
residues on the tissue.  Lectins are highly selective for these mono and
oligosaccharides. 

Consequently, UEA is not naturally occuring IN the tissue, rather detects
the  carbohydrate residue.  You can buy UEA biotinylated, UEA HRP, UEA FITC
and work with this, eliminating a secondary antibody altogether.  

Be sure to use a negative control, go to Vector website, and look at their
lectin selection.  A each on you will see the sugar you need (also the one
UES is detecting in tissue).  This is the inhibiting sugar and to create a
negative control) - you take UEA and/or any of its conjugates and incubate
it for 30 min with the recommended concentration of the sugar, apply this
to section. 

You can buy UEA unconjugated, then detect with whatever conjugate of Rabbit
antiUEA you want, proceed with IFA or IHC. You still need to do the
negative control with UEA itself.

We frequently use UEA-Biotinylated proceed with Strepavidin HRP or AP for
IHC, or apply STrepavidin Alexa 488 OR UEA-FITC alone for fluorescence. 

There is a wonder, VERY inexpensive book from SpringerVerlag, Lectin
HIstochemistry by Brooks, Leathem and Schumacher, ISBN #1 85996 100 2.  It
has a list of lectin binding studies to human and animal tissue, plus
references. I have found it invaluable - immunostaining technics are also
in book.  

Some lectins require a lectin buffer a TBS with magnesium and calcium added
as PBS may not be acceptable for good staining. You can also talk to Craig
Pow at Vector about this, he was very helpful to us explaining buffer and
negative control needs (before we bought lectin book!)

I strongly recommend purchasing this book

 

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu