[Histonet] Re: Lectin IHC

[Barry R Rittman]

John
The lectin binding may also depend on the processing as well as the fixation.  We carried out lectin binding some years ago on tissue fixed in buffered formalin and processed to paraffin wax using chloroform as the intermediary agent. We could not repeat the results on tissue that was sent to us from another lab. The difference was traced to the other laboratory used xylene in their processing schedule instead of chloroform. This would suggest that some the lectins we were examining were binding to glycolipids and that some of these were removed by the xylene.
Barry

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of John C. Dennis
Sent: Friday, April 02, 2004 10:46 AM
To: Jan Shivers
Cc: histonet
Subject: Re: [Histonet] Re: Lectin IHC


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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