Alexa fluor 488 anti rabbit igg secondary antibody

For flow cytometry analysis, the macrophages were seeded onto sterilized immuno-functioned 3D-scaffolds. After 3 days of culture, the rBMDMs were collected and stained with rabbit polyclonal anti-CD206 (Abcam, Cambridge, UK) and mouse monoclonal anti-CD68 (Abcam) for 1 h. Then the cells were centrifuged to remove extra antibodies, and stained with Alexa Fluor 488 anti-rabbit IgG secondary antibody (Abcam) and Alexa Fluor 647 anti-mouse IgG secondary antibody (Abcam) for 30 min. The expression of CD68 and CD206 on the rBMDMs growing on the immuno-functioned 3D-scaffolds was analyzed by a flow cytometer.

Paraffin-embedded samples for immunofluorescence were deparaffinized in the xylene substitute Pro-Par Clearant (Anatech, Ltd., Battle Creek, MI, USA) and rehydrated in graded ethanol and water. After washing with phosphate-buffered saline (PBS), the sections (5-µm) were blocked with 5% goat serum for 1 h at room temperature and then incubated at 4°C with rabbit polyclonal anti-LC3II (1:50; NB910-40435; Sigma-Aldrich, St. Louis, MO, USA) overnight. After further washing with PBS, the sections were incubated with the Alexa Fluor® 488 anti-rabbit IgG secondary antibody (Abcam, Cambridge, MA, USA) for 30 min. Finally, the sections were washed and observed using fluorescence microscopy