Oral Biomedicine ›› 2024, Vol. 15 ›› Issue (1): 12-20.

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Experimental study about the fluorescence imaging of microvesicles based on protein retention expansion microscopy

  

  1. 1. State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration
    2.
    3. Department of Orthodontics, the Third Affiliated Hospital of Air Force Medical University
    4. State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration,
    5. State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, National Clinical Research Center for Oral Diseases, Shaanxi Key Laboratory of Stomatology, Department of Operative Dentistry and Endodontics, the Third Affiliated Hospital of Air Force Medical University
    6. State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, National Clinical Research Center for Oral Diseases, Shaanxi International Joint Research Center for Oral Disease, Center for Tissue Engineering, the Third Affiliated Hospital of Air Force Medical University
    7. Department of Orthodontics, School of Stomatology, The Air Force Military Medical University
  • Received:2023-11-27 Revised:2024-01-11 Online:2024-02-25 Published:2024-03-23

Abstract: Objective:To investigate the compatibility of protein retention expansion microscopy (proExM) with mouse mandibular-derived microvesicles (MVs), and to observe the distributions of surface markers on MVs after expansion, as well as to accurately identify their cellular origins. Methods:MVs from mouse mandibles were isolated by differential centrifugation; Transmission electron microscopy, Western blot and Nanoparticle tracking analysis were used to identify the MVs; Immunofluorescence staining was conducted for the surface proteins CD9, CD63, alkaline phosphatase (ALP) and osteoclast associated receptor (OSCAR) of MVs; The proExM was used to amplify the MVs after immunofluorescence staining; The expansion coefficient was measured, and the morphology and fluorescence staining of MVs before and after expansion were observed by confocal microscopy. Results:The MVs isolated from mouse mandibles by differential centrifugation met the identification criteria for MVs; Under the experimental procedure, a 4-fold expansion of mouse mandibular-derived MVs was achieved by expansion of the gel; The fluorescence intensity distributions of CD9 and CD63 on the line profile of MVs after expansion presented multi-peak patterns; After expansion, the variance of mander’s colocalization coefficient (MCC) 1 of MVs increased significantly (P＜0.01), while the variance of MCC2 decreased (P＜0.05), and the variance of pearson correlation coefficient (PCC) increased significantly (P＜0.01); After expansion, accurate identification of MVs secreted by osteoblasts and osteoclasts was achieved; After expansion, it was measured that osteoblast-derived MVs accounted for 11.11% of the CD9+ MVs derived from mouse mandibles, while osteoclast-derived MVs accounted for 3.70%. Conclusions:This experimental procedure could improve the resolution in the observation of mouse mandibular-derived MVs. In this study, a standard experimental procedure was established to reveal the heterogeneity of surface protein distribution of MVs and to achieve the accurate identification of the cell origins of tissue-derived MVs.

Key words: protein retention expansion microscopy, microvesicles, surface markers, fluorescence colocalization, single vesicle analysis