[Histonet] Re: Lectin IHC

From:"Jan Shivers"

I deleted the initial start of this discussion prematurely, but I wanted to
add that back when I did lectin staining years ago, I found that not all
lectins stained consistently between the various species.  I reasoned that
it was perhaps a variance in the glycoconjugates found on different species'
cells.  The lectin I wanted to work the most (UEA), I could not get to work
on canine endothelium at all, for instance, though I had some success with
other lectins on other cell types.  Of course, I admit that maybe I had just
not found the magic protocol to make the UEA work on dog tissue.  However,
one might consider that species variability could be the reason why one
lectin works on human material but not on guinea pig or other mammals.

Jan Shivers
U of MN Vet Diag Lab

----- Original Message ----- 
From: "Pablo Sánchez Quinteiro" 
Sent: Thursday, April 01, 2004 12:48 PM
Subject: [Histonet] Re: Lectin IHC

Hi Jenny,

Nothing unexpected in your protocol. I usually incubate the UEA-I overnight
at 4ºC. It could make a difference. 15 mins of substrate (DAB?) is a long
time. Staining should come off in a few minutes.

I guess you use biotinylated lectin. With UEA-I I have tried both the
biotinylated lectin + ABC from Sigma or the UEA-I + Peroxidase-linked
antibody against UEA-I (from Dako). This works much better.

You do not tell in what organ or tissue you employs UEA I. That is also
relevant. For example in nervous system UEA-I only stains the olfactory
bulbs. For nervous system you can see P.C. Barber, Neuroscience 30:1-9
(1989). He discusses different fixatives and protocols.



At 10:06 a.m. 01/04/04 -0800, you wrote:
>Hi To all,
>Here is more detailed descripiton of what I have done. The organism I
>am studying is Guinea Pig. The tissues are infiltrated with OCT and kept
>frozen until cut.
>This is the General Protocol of my lectin staining
>1. Air Dry for 15 mins
>2. Application of fixative (depending on the reagents used, incubation
>time changes) and tap water rinse
>3. Block for 30mins (3X Buffer Wash)
>4. Lectin Incubation for 1 hr (3X Buffer Wash)
>5.Application of ABC for 30 mins(3X Buffer Wash)
>6. Application of substrage for 15 mins (tap water rinse)
>7. Counterstain with mayer's hemotoxylin
>The combinations that I have tried are ( fixitive/block/dectection
>10% Formalin/ universal block/ avidin-biotin
>10% formalin/ 0.1% BSA block/avidin-HRP or avidin-Biotin
>Acetone/0.1%BSA block/avidin-HRP
>Bouin/universal block or 0.1% BSA block/avidin-biotin
>I haven't been able to get a positive staining for my positive control.
>The lectin I been using is UEA-1 Any suggestion would be great.

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