[Histonet] Re: Lectin IHC
Pablo Sánchez Quinteiro
psanquin <@t> lugo.usc.es
Thu Apr 1 12:48:44 CST 2004
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
>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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