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Evaluation of human platelet granules by structured illumination laser fluorescence microscopy

Authors :
Fred G. Pluthero
Walter H. A. Kahr
Source :
Platelets, Vol 34, Iss 1 (2023)
Publication Year :
2023
Publisher :
Taylor & Francis Group, 2023.

Abstract

Many roles of human platelets in health and disease are linked to their ability to transport and secrete a variety of small molecules and proteins carried in dense (δ-) and α-granules. Determination of granule number and content is important for diagnosis of platelet disorders and for studies of platelet structure, function, and development. We have optimized methods for detection and localization of platelet proteins via antibody and lectin staining, imaging via structured illumination laser fluorescence microscopy (SIM), and three-dimension (3D) image analysis. The methods were validated via comparison with published studies based on electron microscopy and high-resolution fluorescence microscopy. The α-granule cargo proteins thrombospondin-1 (TSP1), osteonectin (SPARC), fibrinogen (FGN), and Von Willebrand factor (VWF) were localized within the granule lumen, as was the proteoglycan serglycin (SRGN). Colocalization analysis indicates that staining with fluorescently labeled wheat germ agglutinin (WGA) allows detection of α-granules as effectively as immunostaining for cargo proteins, with the advantage of not requiring antibodies. RAB27B was observed to be concentrated at dense granules, allowing them to be counted via visual scoring and object analysis. We present a workflow for counting dense and α-granules via object analysis of 3D SIM images of platelets stained for RAB27B and with WGA. Abbreviation: SIM: structured illumination microscopy; WGA: wheat germ agglutinin; FGN: fibrinogen; TSP1: thrombospondin 1; ER: endoplasmic reticulum

Details

Language :
English
ISSN :
09537104 and 13691635
Volume :
34
Issue :
1
Database :
Directory of Open Access Journals
Journal :
Platelets
Publication Type :
Academic Journal
Accession number :
edsdoj.5394bf09b2b74287987a9baea8a4f692
Document Type :
article
Full Text :
https://doi.org/10.1080/09537104.2022.2157808