Transverse sectioning of plastic-embedded immunolabeled cryosections: morphology and permeability to protein...

In order to provide data for meaningful interpretation and quantitation of immunogold labeling on cryosections their morphology and permeability to protein A-gold were evaluated: We studied plastic sections of immunogold-labeled ultrathin and semithick cryosections cut perpendicular to the original cryosection plane. Various soluble and insoluble antigens in different specimens (hemoglobin and histone H5 in chicken erythrocytes, tubulin in Leishmania cells, and outer membrane protein OmpA in Escherichia coli) were fixed with glutaraldehyde-formaldehyde, formaldehyde, or periodate-lysine-paraformaldehyde and incubated with specific antibodies and protein A-gold of different sizes. The cryosection surface may be rough or smooth depending both on the sectioned material and on dehydration and drying artifacts or possibly on the cutting process itself. Well-preserved sections are capable of withstanding considerable deformation without showing clefts or cracks. If the sectioned specimen is sufficiently fixed, protein A-gold is not able to enter the IgG-labeled sections significantly but follows surface irregularities. However, gold particles can be detected within visibly damaged sections.