Glycerol-based Anti Fade Mediums

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From:"Plummer, Timothy B." <> (by way of histonet)
To:histonet <>
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A Monday Story:

A few months ago I contacted Vector laboratories regarding the observation
that a product of theirs, Vectashield-DAPI was causing very rapid decay or
solubilization of the signal arising from tissue stained with the
FITC-conjugated Isolectin B4 (Griffonia Simplicifolia).  The anti fade media
normally will maintain fluorescent signal for extended periods of time
(weeks), but in the case of IB4, the signal would be very intense after
coverslipping, but within several hours the fluorochrome would dissolve away
from its original binding position and be floating around the area of
reactivity in a diffuse pattern.  I contacted Vector at the time and they
stated that the glycerol in the medium was binding up the lectin and pulling
it back into solution (mounting medium).  They (Vector) recommended diluting
the Vectashield-DAPI 1:8 in PBS, this action would supposedly reduce the
glycerol binding and still maintain some anti fading characteristics.  The
remedy has worked as far as the diffusion is concerned, although the
specimens will develop air bubbles if not sealed with nail polish, but the
anti fading mechanism has not been acceptable.

Since that time, I have noticed this phenomena with anti Smooth Muscle Actin
(clone 1A4, Sigma FITC conj.), and also an in-house MAb directed toward von
willibrand factor (unconjugated).  My questions are:  1. Can anyone explain
this phenomena as described and as to why only certain antibodies and
lectins seem to be affected?
2. Are there anti fade mediums which do not contain glycerol that people
have used successfully?

Based upon discussions with Vector there must be some characteristic of
certain antibodies and lectins that will allow attachment to glycerol or
perhaps some other ingredient within the media.  This effect may only be
seen with procedures using detection methods which do not deposit an
insoluble reaction (such as seen with enzyme-based reactions) and allow the
antibodies to detach or be pulled away from their binding location by
something with a higher affinity or binding strength.

I appreciate all comments and thank you.


Timothy B. Plummer
ImmunPathology Core
Transplantation Biology Research Laboratory
Mayo Foundation
Rochester, MN 55905

Phone:	507 538 0689
Fax:	507 284 4957
Pager:	507 281 6430 (numerical)

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