Experimental study about the fluorescence imaging of microvesicles based on protein retention expansion microscopy
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 i-dentify 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 fluo-rescence 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 proce-dure could improve the resolution in the observation of mouse mandibular-derived MVs.In this study,a standard experimental proce-dure 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.
protein retention expansion microscopymicrovesiclessurface markersfluorescence colocalizationsingle vesicle analysis
万方数据
维普
