cGMP-Immunocytochemistry

Immunocytochemistry (ICC) of small, water-soluble molecules requires some special precautions compared to ICC of large protein molecules. In general, protein antigens are relatively insensitive to the type of fixative used. Apparently, the epitopes are not altered in a decisive way by the fixative, immobilization is usually effective, and more than one eptiope may be recognized by polyclonal antisera. In studying by ICC the location of small, hydrophilic molecules—such as neurotransmitters and cyclic nucleotides—choice of fixative plays a decisive role. First, fixation is necessary not only to preserve tissue morphology, but also to fix the molecules to the tissue matrix, otherwise these may be lost from the tissue. This is certainly the case for cyclic nucleotides (1 –4 ). Second, the fixative may covalently modify the hapten by fixing it to tissue protein. This has been shown to occur with serotonim (5 –6 ) and also with small peptides (7 ). Because the covalent modification of the hapten may be part of the antigenic determinant, it is a prerequisite that antisera are raised against conjugates prepared using a fixative that is also suitable for tissue fixation (8 –10 ).