Preservation of spatial organization and antigenicity of leukocyte surface molecules by aldehyde fixation: flow cytometry and high-resolution FESEM studies of CD62L, CD11b, and Thy-1.
We used transmission and scanning electron microscopy in conjunction with immunogold labeling to study cell surface molecules for evidence of distribution-function relationships. Ascription of functional significance to surface distribution therefore requires preservation of cell morphology and maintenance of molecular expression and distribution through the multiple steps of cell preparation. These requirements prompted us to compare two methods for preparing leukocytes for analysis of surface molecule distribution: one method involved using low temperature to "stabilize" cell morphology and surface molecular organization through immunolabeling; the other involved fixation of the cells with dilute glutaraldehyde before their isolation and labeling. Binding of primary antibodies to several surface molecules, measured by flow cytometry, was comparable for cells prepared by the two methods. Cell morphology and molecular distributions, assessed by high-resolution field emission SEM, were likewise comparable. These results support the conclusion that cell morphologies and CAM distributions previously reported were not affected by exposure of the cells to low temperature through isolation and immunolabeling. Our additional observation that Thy-1 is expressed on both non-projecting and projecting membrane domains of mouse lymph node lymphocytes and rat thymocytes represents a third and new pattern of surface molecule distribution.
Hasslen
S R
SR
Department of Laboratory Medicine and Pathology, University of Minnesota School of Medicine, Minneapolis 55455, USA.
Burns
A R
AR
Simon
S I
SI
Smith
C W
CW
Starr
K
K
Barclay
A N
AN
Michie
S A
SA
Nelson
R D
RD
Erlandsen
S L
SL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions / Q000187:drug effects*; D003080:Cold Temperature; D005404:Fixatives / Q000494:pharmacology*; D005434:Flow Cytometry; D005976:Glutaral / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D019041:L-Selectin / Q000032:analysis*; D007962:Leukocytes / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure*; D008198:Lymph Nodes / Q000166:cytology; D016177:Macrophage-1 Antigen / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D016253:Microscopy, Immunoelectron / Q000379:methods*; D051381:Rats; D011919:Rats, Inbred Strains; D013048:Specimen Handling; D018800:Thy-1 Antigens / Q000032:analysis*; D016707:Tissue Fixation / Q000379:methods*
D000911:Antibodies, Monoclonal; D005404:Fixatives; D016177:Macrophage-1 Antigen; D018800:Thy-1 Antigens; D019041:L-Selectin; D005976:Glutaral