Immunolabelling of bacteriophage lambda receptor protein (LamB) on thin sections of E. coli embedded in Lowicryl.
LamB is one of the major cellular proteins when E. coli is grown in the presence of maltose and is localized in the outer membrane. Previous immunolabellings obtained with monoclonal antibodies showed that this protein is a transmembrane protein and led to the detection of 4 epitopes exposed on the cell surface and 2 located on the inner surface of the outer membrane (Scheckman et al., 1983). In the present study, we have used this biological model in order to see whether these two classes of epitopes could be distinguished by immunocytochemical labelling performed on thin sections of E. coli embedded in Lowicryl K4M (Carleman et al., 1982). The optimal conditions of fixation and embedding were first established for labelling with poly- or monoclonal antibodies detected by Protein A-gold complexes. The analysis of gold particle distribution on each side of the outer membrane after labelling with a polyclonal serum or after its adsorption on intact bacteria allowed us to conclude that the resolution of immunolabelling on thin sections was about 20 nm. The use monoclonal antibodies met with difficulties due mostly to the nonspecific labelling of the cytoplasm. Although this nonospecific labelling was decreased by fixing bacteria with paraformaldehyde alone, only one antibody gave a correct specific labelling after high dilution (1/3000). The gold particle distribution obtained with this antibody confirmed the location on the cell surface of this epitope.
D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000276:immunology*; D010582:Bacteriophage lambda / Q000378:metabolism; D000939:Epitopes; D004926:Escherichia coli / Q000276:immunology*; D005404:Fixatives; D006046:Gold; D018272:Porins; D011991:Receptors, Virus / Q000276:immunology*
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D000939:Epitopes; D005404:Fixatives; D018272:Porins; D011991:Receptors, Virus; C016532:maltoporins; D006046:Gold