Paraffin-embedded samples of human fat tissue were selected for immunohistochemical and immunofluorescence analysis. Samples were deparaffinized with xylene and rehydrated by an increasing ethanol gradient for hematoxylin and eosin (H&E) staining. Target retrieval was performed using the PT Link (DAKO) at pH solution 9.0 (DAKO). Immunohistochemistry staining was performed using a Dako Autostainer Link 48. Primary antibodies used were as follows: rabbit monoclonal ALP (Abcam, 1/200), mouse monoclonal CD73 (Abcam, 1/200), mouse monoclonal CD31 (DAKO, 1/200), and the appropriate EnVision HRP secondary antibody (EnVision HRP rabbit or mouse, DAKO, 1/500) according to the manufacturer’s instruction. Immunofluorescence was performed using a Dako Autostainer Link 48 with the following antibodies: rabbit monoclonal ALP (Abcam, 1/200), mouse monoclonal CD73 (Abcam, 1/200), Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher, 1/200), and Alexa Fluor 546 goat anti-mouse IgG (Thermo Fisher, 1/200) according to the manufacturer’s instruction. Sections were visualized with LEICA DM6600 with a × 20 magnifying objective lens.
Canepa D.D., Casanova E.A., Arvaniti E., Tosevski V., Märsmann S., Eggerschwiler B., Halvachizadeh S., Buschmann J., Barth A.A., Plock J.A., Claassen M., Pape H.C, & Cinelli P. (2021). Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential in human adipose-derived mesenchymal stromal cells by mass cytometry. Stem Cell Research & Therapy, 12, 7.
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