Applicability of using acrylic resins in post-embedding ultrastructural immunolabelling of human neutrophil granule proteins.
In this study, quantitative assessments were carried out, (1) by light microscopy during tissue preparation for electron microscopy and (2) by electron microscopy after on-grid immunogold staining, to determine the suitability of using LR White and Lowicryl K4M thin sections to identify lactoferrin and elastase in the granules of human neutrophil leucocytes. Quantitative assessment of the effect of fixation, dehydration and embedding on the preservation of antigenicity during tissue preparation for electron microscopy, using light microscopic peroxidase anti-peroxidase immunocytochemistry, enabled the selection of preparation conditions that adequately preserved both antigenicity and ultrastructure. OsO4 post-fixation, following primary aldehyde fixation, improved the retention of antigenicity during dehydration and embedding and the preservation of fine structure. Partial rather than complete dehydration retained more of the antigenicity. The efficiency, sensitivity and resolution of immunolabelling and the ultrastructure and quality of sections achieved after embedding in LR White were superior to those obtained after embedding in Lowicryl K4M. Consequently room temperature embedding in LR White following double fixation and partial dehydration is a better and more reliable preparation technique than low-temperature embedding in Lowicryl K4M following single fixation and partial dehydration for localizing lactoferrin and elastase to the specific and primary granules respectively in human neutrophilic granulocytes by the on-grid immunogold staining method.
Mutasa
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Department of Haematological Medicine, Cambridge University Clinical School, UK.
D000180:Acrylic Resins; D001798:Blood Proteins / Q000032:analysis*; D003594:Cytoplasmic Granules / Q000032:analysis / Q000648:ultrastructure*; D006046:Gold; D006651:Histocytochemistry; D006801:Humans; D007202:Indicators and Reagents; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000097:blood; D007951:Leukemia, Myeloid / Q000097:blood; D009504:Neutrophils / Q000032:analysis / Q000648:ultrastructure*; D009195:Peroxidase; D013194:Staining and Labeling
D000180:Acrylic Resins; D001798:Blood Proteins; D007202:Indicators and Reagents; D006046:Gold; D009195:Peroxidase