Image pro premier version 9

After deparaffinized, rehydration and antigen retrieval, tissue sections were cooled at room temperature and rinsed in PBS for 5 minutes. After blocking with a blocking solution (Blocking One Histo; Nacalai Testique, Japan) for 1 h to prevent non-specific binding, the sections were incubated with primary rabbit anti-CD68 antibody (#ab125212) as M1 marker and rabbit anti-CD163 antibody as M2 marker (#ab182422) (Abcam, Cambridge, USA) overnight at a dilution of 1:200 in 5% BSA at 4°C. The sections were subsequently incubated with secondary antibody, Alexa-Fluor 647—conjugated goat anti-rabbit IgG (H+L) cross-adsorbed (A-21244) and Alexa-Fluor 488—conjugated donkey anti-rabbit IgG (H+L) highly cross-adsorbed (A-21206) secondary antibodies (Thermo Scientific, USA) respectively at a dilution of 1:400 in 5% BSA for 1 h at room temperature. Sections were washed with PBS prior and after the incubation of secondary antibodies. After washing, sections were mounted in UltraCruz Mounting Media (DAPI) (Santa Cruz, CA, USA). Finally, the slides were then observed under a fluorescence microscope (Olympus BX61, USA) and photomicrographs were taken. All the intensities of immunofluorescence expressions were quantitatively evaluated by using Image Pro-Premier version 9.1 (Media Cybernetics, Rockville, MD).

Frozen liver tissues were cryosectioned, and sections were immersed in absolute propylene glycol for 2 minutes and stained with Oil Red O for an hour. Then, the sections were differentiated in 85% propylene glycol solution for 1 minute, stained with hematoxylin, differentiated with acid alcohol and neutralized in weak ammonia water, and eventually mounted with glycerin jelly. Images were obtained under bright field LEICA DM 2000 microscope with the utilization of LEICA ICC50 HD camera (Leica Microsystems, Germany) and the hepatic mean intensities of Oil Red O-stained positive areas were quantitatively evaluated by using Image Pro-Premier version 9.1 (Media Cybernetics, Rockville, MD).

Kidney and liver tissue sections were deparaffinized by immersion in xylene and rehydrated through an ethanol series. The sections were then incubated in a microwave in 0.01 M citrate buffer (pH 6.0, 10 min, 95–100°C) for antigen retrieval. To eliminate endogenous peroxidase activity, the sections were incubated with 3% hydrogen peroxide solution (Peroxidazed 1; Biocare Medical, Pacheco, California). Following incubation with a blocking solution (Blocking One Histo; Nacalai Tesque, Inc, Japan) for 1 h to prevent non-specific binding, the sections were incubated with primary rabbit IL-6 (1:200, sc-1265), MCP-1 (1:200, sc-1785) or TNF-α antibodies (1:200, sc-52746, Santa Cruz Biotechnology, California, USA) overnight at 4°C. The sections were subsequently washed and incubated with appropriate HRP conjugated polyclonal biotinylated secondary antibodies (1:400, sc-2004, Santa Cruz Biotechnology, California, USA and #P0447, Dako, Denmark). The sections were visualized with 3,3ʹ-diaminobenzidine tetrahydrochloride (DAB) substrate, counter-stained with hematoxylin and examined by brightfield microscopy (LEICA DM 2000) and images were obtained with a LEICA ICC50 HD camera (Leica Microsystems, Germany). All the immunohistochemical expressions were quantitatively evaluated by using Image Pro-Premier version 9.1 (Media Cybernetics, Rockville, MD).