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

Sharon R. Hasslen, Alan R. Burns, Scott I. Simon, C. Wayne Smith, Karen Starr, A. Neil Barclay, Sara A. Michie, Robert D. Nelson, Stanley L. Erlandsen

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1115-1122
Number of pages8
JournalJournal of Histochemistry and Cytochemistry
Volume44
Issue number10
StatePublished - 1996
Externally publishedYes

Fingerprint

Aldehydes
Flow Cytometry
Leukocytes
Scanning Transmission Electron Microscopy
Temperature
Glutaral
Thymocytes
Lymph Nodes
Maintenance
Lymphocytes
Membranes
Antibodies

Keywords

  • Aldehyde fixation
  • Flow cytometry
  • High-resolution SEM
  • Immunocytochemistry
  • L-Selectin (CD62L)
  • Lymphocyte
  • Neutrophil
  • Thy-1 (CD49d)
  • Thymocyte
  • β2-Integrin (CD11b/CD18)

ASJC Scopus subject areas

  • Anatomy
  • Cell Biology

Cite this

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. / Hasslen, Sharon R.; Burns, Alan R.; Simon, Scott I.; Smith, C. Wayne; Starr, Karen; Barclay, A. Neil; Michie, Sara A.; Nelson, Robert D.; Erlandsen, Stanley L.

In: Journal of Histochemistry and Cytochemistry, Vol. 44, No. 10, 1996, p. 1115-1122.

Research output: Contribution to journalArticle

Hasslen, SR, Burns, AR, Simon, SI, Smith, CW, Starr, K, Barclay, AN, Michie, SA, Nelson, RD & Erlandsen, SL 1996, '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', Journal of Histochemistry and Cytochemistry, vol. 44, no. 10, pp. 1115-1122.
Hasslen, Sharon R. ; Burns, Alan R. ; Simon, Scott I. ; Smith, C. Wayne ; Starr, Karen ; Barclay, A. Neil ; Michie, Sara A. ; Nelson, Robert D. ; Erlandsen, Stanley L. / 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. In: Journal of Histochemistry and Cytochemistry. 1996 ; Vol. 44, No. 10. pp. 1115-1122.
@article{df5d4ab8c96d4861bd7ca04a6c7b11f4,
title = "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",
abstract = "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.",
keywords = "Aldehyde fixation, Flow cytometry, High-resolution SEM, Immunocytochemistry, L-Selectin (CD62L), Lymphocyte, Neutrophil, Thy-1 (CD49d), Thymocyte, β2-Integrin (CD11b/CD18)",
author = "Hasslen, {Sharon R.} and Burns, {Alan R.} and Simon, {Scott I.} and Smith, {C. Wayne} and Karen Starr and Barclay, {A. Neil} and Michie, {Sara A.} and Nelson, {Robert D.} and Erlandsen, {Stanley L.}",
year = "1996",
language = "English (US)",
volume = "44",
pages = "1115--1122",
journal = "Journal of Histochemistry and Cytochemistry",
issn = "0022-1554",
publisher = "Histochemical Society Inc.",
number = "10",

}

TY - JOUR

T1 - Preservation of spatial organization and antigenicity of leukocyte surface molecules by aldehyde fixation

T2 - Flow cytometry and high-resolution FESEM studies of CD62L, CD11b, and Thy-1

AU - Hasslen, Sharon R.

AU - Burns, Alan R.

AU - Simon, Scott I.

AU - Smith, C. Wayne

AU - Starr, Karen

AU - Barclay, A. Neil

AU - Michie, Sara A.

AU - Nelson, Robert D.

AU - Erlandsen, Stanley L.

PY - 1996

Y1 - 1996

N2 - 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.

AB - 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.

KW - Aldehyde fixation

KW - Flow cytometry

KW - High-resolution SEM

KW - Immunocytochemistry

KW - L-Selectin (CD62L)

KW - Lymphocyte

KW - Neutrophil

KW - Thy-1 (CD49d)

KW - Thymocyte

KW - β2-Integrin (CD11b/CD18)

UR - http://www.scopus.com/inward/record.url?scp=0029834571&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029834571&partnerID=8YFLogxK

M3 - Article

VL - 44

SP - 1115

EP - 1122

JO - Journal of Histochemistry and Cytochemistry

JF - Journal of Histochemistry and Cytochemistry

SN - 0022-1554

IS - 10

ER -