Buffers for RE: [Histonet] Using lectin BS-1 for staining endothelial cells

[Gayle Callis]

Ray is correct on the problem of using PBS with lectins. After much 
discussion with Vector's Craig Pow (Technical service representative), he 
indicated not ALL lectins are affected by PBS. We have had successful 
staining with UEA1 and PNA using Dulbeccos PBS.  More recently, we use TRIS 
buffer exclusively for all lectin staining.   There is also  "Lectin 
Buffer" that is TRIS based on the chance you do not have HEPES buffer.  See 
recipe below:

TRIS base               60.57 g
sodium chloride        87 g
Magnesium chloride   2.03g
calcium chloride         1.11g
Dissolve in  800 mls distilled water.  Adjust pH to 7.6 with concentrated 
HCl, then bring to final volume of 1 liter.  This is a 10X solution, can be 
stored in the refrigerator, and diluted 1:10 when needed.  Or you can bring 
this 0ne liter of 10X to a final volume of 10 liter using distilled water.

What you have is a popular immunostaining buffer,  0.05M TBS buffer but 
with the Mg and Ca added.

The lectin buffer comes from an inexpensive, excellent book titled Lectin 
Histochemistry, a concise practical handbook  Brooks SA, Leathem AJC, 
Schumacher U.  ISBN# 1859961002 .

Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717




t 02:18 PM 10/3/2006, you wrote:
>Gabriel,
>It is always hard to come up with optimal solution to any staining problem
>via internet since there are so many variables in procedure.  One you might
>consider, if you haven't already, is the buffer used in your staining
>diluent.  For biotinylated lectins, we always use HEPES buffer (not PBS).
>Many lectins require Mg++ or Ca++ to bind optimally and such ions will
>precipitate out in PBS.  In fact if you look at the Vector website, under
>information for biotinylated Griffonia (their genus name for Bandieraea)
>simplicifola I, it states that "binding appears to require divalent cations
>such as calcium and magnesium".
>
>Ray
>Raymond Koelling
>Research Scientist, Pathology
>Amgen Corp.
>Seattle, WA
>
>-----Original Message-----
>From: histonet-bounces <@t> lists.utsouthwestern.edu
>[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gabriel
>Anesetti
>Sent: Monday, October 02, 2006 3:51 PM
>To: histonet <@t> lists.utsouthwestern.edu
>Subject: [Histonet] Using lectin BS-1 for staining endothelial cells
>
>Hello Histonetteers,
>I am having problems with the staining of rat endothelial cells in
>paraffin sections using a biotin labeled lectin from Bandeiraea
>Simplicifolia BS-1 (from Sigma). The lectin does not appear to be
>staining any of the endothelial cells. I have tried using citrate buffer
>for antigen retrieval and DAB, DAB + NiCl or FICT to visualize the site
>of reaction. Sections are from ovary grafts and have very new blood
>vessels forming. Any information would be helpful.
>