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A comparative study for ultrastructural localization of intracellular immunoglobulins using peroxidase conjugates

Authors
Journal
Journal of Immunological Methods
0022-1759
Publisher
Elsevier
Publication Date
Volume
5
Issue
1
Identifiers
DOI: 10.1016/0022-1759(74)90043-x
Disciplines
  • Biology

Abstract

Abstract Various experimental conditions were tried to stain intracellular IgG globulins within popliteal lymph node cells of rats and rabbits by the use of specific antibodies and their Fab fragments labelled with peroxidase following two different procedures. Fixation with 4% formaldehyde/24 hr, 1% formaldehyde and 1% glutaraldehyde/1–1.5 hr and 1–1.5% glutaraldehyde/1–1.5 hr provided satisfactory ultrastructural conservation of the tissues. None of these fixatives appeared to denature IgG globulins, but formation of cross-linkages prevents conjugates from reaching antigenic sites. Weak aldehyde fixation may enhance penetration of conjugates; however, ultrastructural detail is poorly preserved and proteins will leak out from insufficiently fixed tissue, thus increasing the possibility of staining artifac Under our conditions, best results were obtained by using frozen sections prepared from well-fixed specimens. Apparently, the most important points are tissue fixation and sampling, above all other experimental steps. Conjugates with molar ratios of antibody or Fab fragment to peroxidase being one, seem more effective, although no significant differences were noted between peroxidase conjugates of whole antibody molecules and Fab fragments.

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