Glutaraldehyde protein-antibody fixative

Fixation of proteins to the membrane can be used to prevent their elution during washing and incubation steps. Some methods have been tried (35,80), but epitopes are sensitive to this treatment and may no longer be detectable by antibody. Another method is to fix the antibody-antigen complexes to the nitrocellulose membrane with glutaraldehyde, after they had been formed (81). 

Fig. 7. Section of a spinach leaf, fixed in glutaraldehyde and embedded in K4 M resin, which has been treated with rabbit antibody to the 68 kDa apoprotein of the P-700-Chl a-protein of the PS II complex followed by goat anti-rabbit antibody with 20-nm gold particles attached (see Refs. 84 and 88) (Good-child, D.J. and Anderson, J.M., unpublished results).

Fixed embryos or tissues expressing GFP can also be labeled with antibodies to other proteins or with DNA stains or other compounds that bind specific cellular components, e.g., rhodamine-phalloidin to label F-actin (Wang and Hazelri 1994 Breitweiser et al. 1996 Edwards et al. 1997 Brand 1999). In our laboratory, we find that GFP s fluorescence is destroyed by extended exposure to methanol, so we omit steps that require methanol but others report that short exposures to methanol are acceptable (Brand 1999). The methods given below (Protocols 17.6-17.8) all use formaldehyde fixation, but glutaraldehyde fixation also preserves GFP s fluorescence (Ward 1998). 

One of the two protocols presented here has been adjusted to process paraformaldehyde-fixed tissues while the second for tissues that require glutaraldehyde fixation since the primary antibodies utilized are developed with immunogens obtained by coupling small molecules (e.g., neurotransmitters) with carrier proteins (for details see AO). The techniques described herein are based on those... 

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