Alex fluor 488 labeled secondary antibody

Cells were cultured on glass coverslips in the DMEM based medium. The cultures were treated with LPS incorporating with ENERGI-F704 or compound C in indicated concentrations for 1 h at 37°C. The cells were fixed with 4% paraformaldehyde and subsequently incubated with anti-p65 antibody (1 : 200, number 8242, cell Signaling Technology) at 4°C overnight followed by the incubation with Alex Fluor 488-labeled secondary antibody (1 : 1000, Abcam, Cambridge, UK) for 1 h at room temperature. The nuclei of cells were counterstained with DAPI. Coverslips were mounted with ibidi mounting medium (ibidi GmbH, Martinsried, Germany) and the immunofluorescence was imaged by OLYMUS 1X71 inverted microscope. The resulting images were quantified using ImageJ software.

The sections were dewaxed, rehydrated, and then immersed into citrate buffer for antigen repair. After that, the sections were blocked with 2% bovine serum albumin and then incubated overnight at 4 °C with the following primary antibodies: anti-iNOS (1:200; Invitrogen, USA), anti-ionized calcium binding adaptor molecule-1 (Iba-1) (1:100, Abcam, USA), anti-Arg-1 (1:200, Santa Cruz Biotechnology, USA), followed by incubation with the secondary antibodies Alex Fluor® 488-labeled secondary antibody (green; 1:1000; Abcam), Alex Fluor® 647-labeled secondary antibody (red; 1:1000; Abcam) and Hoechst (blue; 1:1000) for 1 h at room temperature. Slides were observed by a fluorescent microscope (Olympus IX-71) equipped with a Canon EOS digital camera. The iNOS, Arg-1, and Iba-1 positive cells were separately counted using Image J software.

The sections were dewaxed, rehydrated, and then immersed into citrate buffer for antigen retrieval. After that, the sections were blocked with 2% bovine serum albumin and then incubated overnight at 4 °C with the following primary antibodies: anti-ionized calcium binding adaptor molecule-1 (Iba-1) (1:100; Abcam, Cambridge, MA, USA), anti-CD68 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-CD206 (1:200; Santa Cruz Biotechnology), anti-P2X7 (1:200; Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA), anti- microtubule-associated protein 2 (MAP2; 1:200; Abcam), anti-CD11b (1:200; Abcam), followed by incubation with the secondary antibodies Alex Fluor® 488-labeled secondary antibody (green; 1:1000; Abcam), Alex Fluor® 647-labeled secondary antibody (red; 1:1000; Abcam) and DAPI (blue; 1:1000; Santa Cruz Biotechnology) for 1 h at room temperature. Slides were observed by a fluorescent microscope equipped with a Canon EOS digital camera.