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Optimal techniques for the immunocytochemical demonstration ofβ-thromboglobulin, platelet factor 4, and fibrinogen in the alpha granules of unstimulated platelets

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Summary

The distribution of β-thromboglobulin, platelet factor 4, and fibrinogen in unstimulated platelets was investigated by several immunocytochemical techniques. All three substances were found to be localized in the majority of platelet alpha granules either by immunoperoxidase methods on saponin-treated platelets or by colloidal gold immunoconjugates on frozen thin sections. The optimal conditions for preparing and fixing platelets for immunocytochemistry were also determined. Platelets obtained from blood dripped directly into fixative or anticoagulated blood were compared systematically with respect to shape. Temperature was found to be the most important variable. Immediately fixed platelets were generally disc-shaped, regardless of the temperature of the fixative. Reducing the temperature of blood (stored with anticoagulant) before fixation resulted in more swollen and fewer disc-shaped platelets. However, if the blood was mixed with an anticoagulant and maintained at 37° C for 1 h before fixation, the same number of disc-shaped platelets were present as in samples from blood fixed immediately. The intracellular localization of β-thromboglobulin, platelet factor 4, and fibrinogen was consistent regardless of platelet preparatory procedure, but several technical problems were encountered with respect to plasma membrane labelling when control experiments were analysed. Immediately fixed, non-permeabilized platelet plasma membranes were always labelled, no matter which control substances or immunoperoxidase markers were used. However, when platelets were washed by centrifugation, the plasma membranes were negative. Exposure to saponin markedly diminished labelling of the plasma membranes. Optimal techniques for the immunocytochemical demonstration of these alpha granule proteins in platelets are presented in this report.

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Authors and Affiliations

  1. Departments of Pathology and Medicine, University of California School of Medicine, 94143, San Francisco, California, USA

    Paula E. Stenberg, Marc A. Shuman & Dorothy F. Bainton

  2. Department of Medicine, University of Texas Health Science Center, Audie L. Murphy Veterans Hospital, 78284, San Antonio, Texas, USA

    Shirley P. Levine

Authors
  1. Paula E. Stenberg
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  2. Marc A. Shuman
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  3. Shirley P. Levine
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  4. Dorothy F. Bainton
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Additional information

This paper is based in part on theHistochemical Journal Lecture for 1983 given by Dr Bainton at a ‘Symposium on Haematological Cytochemistry’ in Cambridge on 29 September 1983 at the invitation of the Royal Microscopical Society.

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Stenberg, P.E., Shuman, M.A., Levine, S.P. et al. Optimal techniques for the immunocytochemical demonstration ofβ-thromboglobulin, platelet factor 4, and fibrinogen in the alpha granules of unstimulated platelets. Histochem J 16, 983–1001 (1984). https://doi.org/10.1007/BF01003853

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