Re: Re: glutaraldehyde

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Tim Morken wrote as an answer to Scott Stickles question:
>Supposedly glutaraldehyde crosslinks more than formalin and so tissues
>fixed in it should show less immunoreactivity. Even so, electron
>microscopists have no problem immuno-labeling their glut-fixed, resin
>sections. So I would say it won't cause any problems IF it has not been
>fixed too long (will cause same problems as over-fixing in formalin).
Dear Tom and Scott:
Electron microscopists only get good labeling on glutaraldehyde fixed resin
embedded tissue when they are using an antibody that is reacting with
epitopes that are "not" altered by the pretreatment methods that are being
used, i.e. fixation and embedding, and when the antigen can still be
reached. From antibody to antibody the optimal fixation protocol and
embedding procedure might be different and has to be tested.
Both glutaraldehyde and formaldehyde react with protonable amino groups.
However cross-linking as such (is the antigen available for binding) of
glutaraldehyde is not the only difference.  The specific binding of an
antibody to an antigen is mainly dependant on the arrangement of charged
groups in the antigen. Cross-linking  has a stronger effect on the native
charges present in the antigen that is being fixed. Binding of formaldehyde
to a protonable aminogroup results in the formation of a Schiff's base.
Glutaraldehyde does the same but when both aldehyde groups of the
glutaraldehyde molecule bind this results in a resonance stabilized
Schiff's base. This has another effect on the arrangement of charges in the
antigen compared to what you get when using formaldehyde and thus  might
affect antigenicity. If you want to know more about the chemistry behind
fixation with aldehydes the following references in literature might be of
Hopwood, Histochem. 17  (1981), 389
Wold, In:Methods in Enzymology Vol. 25 (1972), 623
Puchtler et al., Histochem. 82 (1985), 201
Fox et al., Histochem. Cytochem. 33 (1985), 845
Hope this is of interest.
Regards, Peter

Peter van de Plas
Costerweg 5
6702 AA Wageningen
The Netherlands
phone: (31)-317-497676
fax: (31)-317-415955

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