Heparin is a potent anticoagulant which can be immobilized on biomaterial surfaces to increase their hemocompatability. In the present work, we have electrochemically synthesized composites comprising heparin and the electrically conducting polymer polypyrrole. The incorporation and exposure of heparin were controlled by varying key conditions of polymer synthesis (i.e., applied current and synthesis time). The resulting composite polymers were electroactive after synthesis and the amount of heparin exposed in the polymer could be increased (up to threefold) by switching the polymers from their oxidized to reduced states. Polymer reduction was achieved by either application of negative potentials (-0.4 to -0.7 V for 90 s) or exposure to aqueous reductant (0.1M sodium dithionite for 30 min). Heparin-polypyrrole composites remained stable after autoclaving, displaying no significant loss of electroactivity, and had a shelf life of at least 2 years postautoclaving. Finally, the composites were found to be excellent substrates for the growth of human endothelial cells.

Two methods are described for the immunocytochemical demonstration of immunoreactive gamma-aminobutyric acid (GABA) in the visual cortex of the cat, an area that contains several types of GABAergic neurons and requires combined methods for their characterization. The first method is illustrated by a representative example of a Golgi-impregnated and gold-toned interneuron of the "bitufted" type situated in layer VI and having an ascending axon. After recording the three-dimensional features of the cell, semithin (0.5 micron) sections of the perikaryon were cut and GABA was demonstrated in the cell body by the unlabeled antibody enzyme method. While immunocytochemistry was used to determine the probable transmitter of the neuron, Golgi-impregnation of the same cell was used to identify its neuronal type. Since aldehyde-osmium fixation was used, further electron microscopic (EM) analysis of the neuron's synaptic connections was possible. The second procedure demonstrated GABA in EM sections of aldehyde-osmium-fixed cortex using protein A-gold as an immunocytochemical marker. Immunoreactivity was found in certain neurons, dendrites, axons, and boutons forming type II synaptic contacts that from previous studies have been thought to be GABAergic. Thus ultrastructural analysis using optimal conditions can now be supplemented with the identification of the transmitter in the same section.

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Antisera to the amino acid gamma-aminobutyric acid (GABA) have been developed with the aim of immunohistochemical visualization of neurons that use it as a neurotransmitter. GABA bound to bovine serum albumin was the immunogen. The reactivities of the sera to GABA and a variety of structurally related compounds were tested by coupling these compounds to nitrocellulose paper activated with polylysine and glutaraldehyde and incubating the paper with the unlabeled antibody enzyme method, thus simulating immunohistochemistry of tissue sections. The antisera did not react with L-glutamate, L-aspartate, D-aspartate, glycine, taurine, L-glutamine, L-lysine, L-threonine, L-alanine, alpha-aminobutyrate, beta-aminobutyrate, putrescine, or delta-aminolevulinate. There was cross-reaction with gamma-amino-beta-hydroxybutyrate, 1-10%, and the homologues of GABA: beta-alanine, 1-10%, delta-aminovalerate, approximately 10%, and epsilon-amino-caproate, approximately 10%. The antisera reacted slightly with the dipeptide gamma-aminobutyrylleucine, but not carnosine or homocarnosine. Immunostaining of GABA was completely abolished by adsorption of the sera to GABA coupled to polyacrylamide beads by glutaraldehyde. The immunohistochemical model is simple, amino acids and peptides are bound in the same way as in aldehyde-fixed tissue and, in contrast to radioimmunoassay, it uses an immunohistochemical detection system. This method has enabled us to define the high specificity of anti-GABA sera and to use them in some novel ways. The model should prove useful in assessing the specificity of other antisera.