Re: Glycerol-based Anti Fade Mediums

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From:Barry Rittman <> (by way of histonet)
To:histonet <>
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                I have also seen this. In my work I found that in general, most
lectins, especially if coupled to FITC, tended to leach out from the mounting
medium often showing a streaming effect. This was much more noticeable with
lectins such as Concanavlin A than with others such as UEA -I, and
more noticeable than with any antibodies that were used. I think that in the
ones that I used the leaching was greatest when there was a high FITC to
ration.  Your comments re binding to glycerol would suggest that the binding to
the antigens in the tissue are weak and bonds can be easily broken in whcih
this probably also occurs with materials other than glycerol.
The bubbles that you saw are true of most glycerol based mounting media unless
sealed and sometimes even then they arise. It can be reduced somewhat by
removing  "embedded bubbles" already present in the medium using an ultrasonic
bath  for 2-5 minutes. The bubble rise to the surface of the medium and

> 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.
> Tim
> 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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