Alexa fluor 594 conjugated donkey anti mouse igg

In the first day after operation, the M0 macrophages were seeded in 24-well plates (25,000 cells per well). After overnight culture, the four doses of BMP-2/CPC particles were placed in upper chamber of Transwell system, and co-cultured for 3 d with M0 macrophages from the second day. Then, the macrophages in the lower chambers were fixed with 4% polyformaldehyde at room temperature for 15 min, and washed with PBS for following immunofluorescent staining. The prepared samples were blocked with 5% donkey serum for 30 min at room temperature, and then incubated with primary antibodies overnight at 4 °C. Alexa Fluor® 488-conjugated Donkey anti-rabbit IgG (1:200) for iNOS, Donkey anti-goat IgG (1:200) for MMR and Alexa Fluor® 594-conjugated Donkey anti-mouse IgG (1:200) for CD68 were purchased from Invitrogen (USA). The cell nuclei were stained by DAPI (Sigma, USA) for 5 min at room temperature. The iNOS- and MMR-positive cells were investigated by a fluorescent microscope (Olympus Corporation, Japan) and analyzed with ImageJ software (National Institutes of Health, USA). The primary antibodies for CD68 (1:100, ab955), iNOS (1:100 ab15323) were purchased from Abcam (UK). The primary antibodies for MMR (1:100, AF2535) was purchased from R&D system (USA).