[Histonet] Re: Lectin IHC

[John C. Dennis]

Jan

What fixative(s) did you use?  In fact, my question deals with UEA in
primates.  The signal is strong but I was dinged for using buffered
(para)formaldehyde.  From what I'm gathering, choice of fixative depends
on what lectin one is interested in?

John Carroll Dennis
Anatomy, Physiology, and Pharmacology
109 Greene Hall
Auburn University, AL  36849


On Thu, 1 Apr 2004, Jan Shivers wrote:

> 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" <psanquin <@t> lugo.usc.es>
> To: <Histonet <@t> lists.utsouthwestern.edu>
> 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.
>
> Regards
>
> Pablo
>
>
> 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
> >method)
> >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.
> >Jenny
> >
> >
>
>
>
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