关键词: 蛋白质保留膨胀显微镜, 微囊泡, 表面标志物, 荧光共定位, 单囊泡分析

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