Preparation and use of recombinant protein G—gold complexes as markers in double labelling immunocytochemistry

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Abstract
Summary Recombinant protein G (RPG) was conjugated to colloidal gold particles and used for immunocytochemistry. In this report, the preparation of RPG—gold conjugates (RPGG) and the application of these conjugates in spot blot tests and in double immunolabelling are described. The immunolabelling was performed on ultracryosections of pig small intestine using antibodies directed against aminopeptidase N and sucrase—isomaltase. The labelling efficiency of RPGG was compared to that of protein A—gold conjugates (PAG) in different compartments of the enterocyte. Quantification showed that the labelling intensity was dependent on the size of the marker as well as on the kind of protein used for complex formation. The distributions for RPGG and PAG were respectively: for the 12nm particles, 10.3 and 6.2 particles/µm of length of microvillar membrane, 3.5 and 1.0 particles/µm2 of Golgi profile and 5.9 and 2.0 particles/µm2 of multivesicular body profile; and for the 6nm particles, 49.6 and 15.7 particles/µm of length of microvillar membrane, 24.4 and 5.0 particles/µm2 of Golgi profile and 25.4 and 3.4. particles/µm2 of multivesicular body profile. Controls showed very little non-specific gold labelling (µm2 of section).