gcallis <@t> montana.edu
Fri Aug 25 13:03:28 CDT 2006
What you want to do is NOT immunostaining but binding the Isolectin-BF4 to
the sugar residues in the rat endothelial cells. For a negative control
you need to use the inhibiting sugar Inhibiting Sugar: 500 mM galactose or
100 mM raffinose and incubate the working concentration of isolectin-B4 in
this at RT for 30 min to one hour, even overnight, and apply to the tissue
section. PBS is NOT a negative control for lectins.
Isolectin-B4 if NOT an antibody so this is NOT immunostaining.
Be sure to use TBS and not PBS as the buffer and no serums in the reagents
since lectins can bind to sugar residues in serums. We simply use TBS
with 0.05% Tween 20, and for fluorescence, reduce the Tween to 0.025% just
so it acts as a sheeting agent for good flow over the tissue. BSA is ok to
use. Some lectins require lectin buffer that is basically TBS with Mg and
Ca added. This should be posted in Histonet Archives.
With fresh snap frozen, in our case, murine tissue, we use 75%acetone/25%
absolute ethanol for 5 min at RT, and go directly to buffer, no more
drying after fixation. I am not sure what methanol will do as a fixative
for lectins, but one can also do formalin fixed paraffin tissue, followed
by trypsin digestion, use isolectin B4- biotinylated and come back with
either Strepavidin-enzyme, then chromogen OR Strepavidin-Alexa fluor for
flourescence work. For frozen sections, lectin incubation is adequate at
30 min, 1 hr for FFPE tissue sections.
Many lectins are conjugated to fluorescein or TRITC (Vector has your lectin
fluoresceinated, and if you do fresh frozen sections, fixed with acetone,
or acetone alcohol, the isolectin B4-fluoresceine conjugate should work
well at around a 1:250 dilution of the 0.5mg concentration.
Do NOT use ABC with this method, no secondaries are needed unless you use
the isolectin B4 unconjugated, then you can detect with an antibody that is
anti-isolectin B4 (if you can find this??)
Try either the direct FITC conjugate or biotinylated conjugate first. The
FITC may conflict with any autofluorescence induced by formalin fixation,
but one could use the biotin and come back with either chromogen method or
red fluorophore conjugated to Strepavdin (Molecular Probes, SA 546, SA-488
is the FITC equivalent). For biotinylated lectin, we buy our Strepavidin
HRP or AP from Biosource, Southern Biotechnology has good SA conjugates as
does Jackson Immunoresearch.
Be aware the lectins can bind to other sites in tissues, if the sugar
residue it recognizes is elsewhere, and there is nothing that blocks this
binding. We have had to do two lectins so see colocalization in order to
sort out what is separate staining elsewhere for a single lectin -
There is a whole book on lectins that is fantastic. Lectin Histochemistry,
a concise practical handbook, Brooks SA and Leathem AJC. ISBN # 1859961002.
Go to Journal of Histochemistry Cytochemistry and pick up the publications
on lectins, one is like a review and tells what lectins bind where, superb!
For help on how to work with lectins, ask Vectors tech rep Craig Pow
(cspow <@t> vectorlabs.com) - he helped me understand a great deal about lectins
and explains why you use inhibiting sugar as the negative control plus
buffer issues, what to avoid. Nice fellow to visit with.
If you want to do immunostaining for rat endothelial cells, you can always
search BD Pharmingen or SEROTEC websites for a monoclonal antibody and do
either true IHC or IFA work.
At 11:25 AM 8/25/2006, you wrote:
>Hello everyone in histoland!
>I finally need to utilze the expertise of all of you fellow histonetters.
>I have limited experience in immunohistochemistry (give me a protocol and
>show me the reagents and I can follow). I am now in a position where I
>will have to work out protocols on my own (oh no! I will have to think!)
>Here is the first assignment. I need to work up a protocol using
>isolectin B4 as a marker for endothelial cells in rat tissue. Would one
>follow the application of the isolectin with an ABC reagent or, as someone
>suggested to me, use a streptavidin derivitive? Does one need to begin
>with an antigen retreivel? I was told that working with lectins can be
>tricky and are considered by some to be "old school" methods. Are there
>better, newer markers or methods for endothelial cells in rat tissue? I
>also have a reference paper that states that 100% methanol was used as a
>fixative. Why use 100% methanol? Is 24 hr fixation in NBF acceptable
>(or preferred)? Any tips, comments, opinions, protocol sharing to lead
>me down the successful lectin path would be appreciated.
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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