After fixing cultured cells with 4% paraformaldehyde for 30 min, the cells were washed with phosphate-buffered saline (PBS) and later incubated with primary antibodies against polyclonal rabbit anti-TSP-1 and TSP-2 (1 : 100; Chemicon, Euromedex, Souffelweyersheim, France), anti-CD38 (1 : 100; Santa Cruz Biotechnology), anti-Akt and p-Akt (1 : 500; R&D Systems, Minneapolis, MN, USA), anti-arginase-1 (Arg-1, 1 : 500; R&D Systems, Minneapolis, MN, USA), anti-formyl peptide receptor 2 (Fpr-2, 1 : 500; R&D Systems, Minneapolis, MN, USA), anti-active caspase-3 (1 : 100; Santa Cruz Biotechnology, Santa Cruz, CA), and anti-early growth response protein 2 (EGR2, 1 : 200; Cayman Chemical, Ann Arbor, MI, USA), as described previously [12 (link)]. Omitting primary antibody controls was used to confirm the specificity of staining signals. Five areas from each slide were randomly selected for image acquisition under 200x magnification. The red immunoreactive signal indicated expression of each specific protein, while the green signal indicated TSP-1 and TSP-2 expression; the Hoechst 33342 blue signal indicated nuclei staining.
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