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

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 12 nm particles, 10.3 and 6.2 particles/micron of length of microvillar membrane, 3.5 and 1.0 particles/micron2 of Golgi profile and 5.9 and 2.0 particles/micron2 of multivesicular body profile; and for the 6 nm particles, 49.6 and 15.7 particles/micron of length of microvillar membrane, 24.4 and 5.0 particles/micron2 of Golgi profile and 25.4 and 3.4 particles/micron2 of multivesicular body profile. Controls showed very little non-specific gold labelling (less than 0.02 gold particles/micron2 of section).