Confocal fluorescence microscopy

The tissues slices were incubated with a polyclonal rabbit anti-IL-10 (bought from Abcam Company, Britain) and mouse monoclonal anti-CD19 (bought from Abcam Company, Britain) antibodies for 16 hours under 4°C at a dilution of 1:300 and 1:100, respectively. Subsequently, the slides were incubated with a mixture of secondary antibody for IF. Both secondary antibodies were used at a dilution of 1:100. Then, CD19/IL-10 Double immunofluorescence staining was observed under Confocal fluorescence microscopy (Olympus, Center Valley, PA, USA), and we performed semi-quantitative analysis by Software Tissuequest 4.0.1.0140. We stained the nucleus with DAPI, spot number of CD19 and IL-10 was collected by the software and DAPI spots number was seemed as reference. We calculated the spots number of CD19 and IL-10/spots number of DAPI, so as to obtain the relative cell counts of CD19 and IL-10 in thymus tissue of different groups.

Differentiated 3T3-L1 adipocytes electroporated with siRNA or plasmid along with pGLUT4-myc-green fluorescent protein (GFP) were grown on coverslips. Twenty-four hours after electroporation, cells were serum-starved for 4 h and treated with or without insulin for 20 min. Cells were then fixed using 4% paraformaldehyde for 10 min without permeabilization, blocked with 3% bovine serum albumin for 1 h at room temperature. Coverslips were immunostained with anti-Myc antibody for 1 h at 37 °C. After incubation, the coverslips were washed three times with PBS solution, followed by incubation with Alexa Fluor 594-labeled goat anti-mouse IgG for 1 h at 37 °C. Coverslips were again washed three times with PBS and mounted in VECTASHIELD medium. PM localization was determined by using confocal fluorescence microscopy (OLYMPUS, Tokyo, Japan) to score 20 representative cells per condition for the appearance of a PM ring of GLUT4. The ratio of GLUT4 translocation was calculated by myc fluorescence on PM/GFP fluorescence in whole cells.

The frozen kidney sections or cell samples were fixed, then blocked and incubated with primary antibodies (LRH‐1: 1:100, GLS2: 1:100) overnight at 4°C, followed by incubation with fluorescent secondary antibodies at 37°C for 90 min in the dark. The nuclei were counterstained with 4, 6‐diamidino‐2‐phenylindole (DAPI, Antgene, China) for 5 min. All microscopic images were detected by confocal fluorescence microscopy (Olympus, Japan).

Primary rat osteoblasts were grown on glass coverslips and incubated with vaspin for 72 h. The cells were washed with cold PBS twice, fixed in ice-cold methanol and permeabilized with 0.5% Triton X-100 for 15 min. BSA was used to block nonspecific binding sites. Samples were then incubated with anti-Runx2 antibody (1:500, Abcam, Cambridge, MA, USA) overnight at 4 °C followed by incubation with tetramethylrhodamine (TRITC)-conjugated secondary antibody and 4′,6-diamidino-2-phenylindole (DAPI). The samples were mounted and observed under a confocal fluorescence microscopy (Olympus, Tokyo, Japan).