Thy-1 immunolabeled thymocyte microdomains studied with the atomic force microscope and the electron microscope.
The atomic force microscope (AFM) and the transmission electron microscope (TEM) have been used to study the morphology of isolated mouse thymocyte microdomains and Thy-1 antigen distribution at the surface of these structures. AFM images were recorded in air in the contact mode on membrane vesicles deposited on previously heated tissue culture plastic sheets and indirectly immunolabeled for Thy-1 expression with colloidal gold-conjugated secondary antibodies. AFM images of untreated plastic plates showed a very characteristic network of streaks 20-200 nm wide. Heating the plastic removed the streaks and provided flat surfaces (r.m.s. 1 nm). This substrate allowed strong adsorption and homogeneous spreading of the vesicles and easy manipulations during immunolabeling experiments. Vesicles flattened on the substrate without losing their morphology. The 10-nm membrane-bound gold beads were reproducibly imaged without degradation by repeated tip scanning. The observed microdomains had a mean diameter of 184 +/- 76 nm, and 65% of them were specifically labeled. Images obtained with the TEM on the same vesicles, deposited on carbon-coated grids and negatively stained, confirmed the AFM observations. The size distribution of the microdomains was quite similar, but the number of beads per vesicle was significantly higher, and 76% of the vesicles were labeled. The difference may be explained 1) by removal of beads from the vesicles in the additional washing step with water, which was necessary for the AFM; 2) by tip-sample convolution; and 3) by statistical fluctuations.
Thimonier
J
J
Centre National de la Recherche Scientifique GDR 976, Parc Scientifique et Technologique de Luminy, Marseille, France. rocca@ciml.univ-mrs.fr
Montixi
C
C
Chauvin
J P
JP
He
H T
HT
Rocca-Serra
J
J
Barbet
J
J
D000818:Animals; D000906:Antibodies; D002462:Cell Membrane / Q000276:immunology / Q000648:ultrastructure; D006046:Gold; D051379:Mice; D008815:Mice, Inbred Strains; D018625:Microscopy, Atomic Force / Q000379:methods; D016253:Microscopy, Immunoelectron / Q000379:methods; D013601:T-Lymphocytes / Q000276:immunology / Q000648:ultrastructure*; D018800:Thy-1 Antigens / Q000032:analysis* / Q000648:ultrastructure*
D000906:Antibodies; D018800:Thy-1 Antigens; D006046:Gold