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The fixed samples were washed in PBS and then transferred stepwise to 100% methanol (MeOH) and stored at ?20?��C. The immunostaining was performed according to the previous report [19] as follows. http://www.selleckchem.com/products/Adriamycin.html The right hepatic lobe was immersed in 15% H2O2, 16.7% DMSO solution in MeOH for 24?h to bleach pigmented cells and to reduce auto fluorescence. The liver then was washed in MeOH, which was repeated five times and then kept at ?80?��C for at least 1?h before return to room temperature. The organ was rehydrated by Tris Buffered Saline-TritonX (TBST) [0.15?m NaCl (Nacalai Tesque, Kyoto, Japan), 0.1?m Tris (hydroxymethy)aminomethane (Nacalai Tesque, Kyoto, Japan) pH 7.4, and 0.1% Triton X-100 (Nacalai Tesque, Kyoto, Japan)]. TBST containing 10% normal goat serum (Dako Corp., Glostrup, Denmark) and 0.01% sodium azide (Nacalai Tesque, Kyoto, Japan) was used as blocking solution for 24?h. The organ was incubated in insulin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in 5% DMSO containing blocking solution for 48?h. After washing, Alexa594 goat anti rabbit IgG (Invitrogen, Carlsbad, CA, USA) was used as secondary antibody for 48?h. For the observations, the immunostained liver was embedded in http://www.selleck.cn/products/MK-1775.html 1% agarose gel (low melting point agarose; Sigma Aldrich, St. Louis, MO, USA) to fix the sample. OPT was performed using an OPT scanner (OPT scanner 3001; Bioptonics, Scotland, UK) according to the manufacturer��s instructions [16,19]. The specimens were maintained within the BABB (benzyl alochol/benzyl benzoate 1:2 ratio), rotated to a series of angular positions (0.9�� apart) and images were captured at each orientation. High-resolutional tomographic images were reconstructed from raw images by NRecon software (SKYSCAN, Kontich, http://www.selleckchem.com/PD-1-PD-L1.html Belgium). The tomographic images obtained from OPT were reconstructed to three-dimensional form and analyzed by Avizo software (Visualization Science Group, Inc., Burlington, MA, USA). Three-dimensional images of islets and liver were obtained by isosurface treatment. Total volume of all islets was calculated by summation of the selected islets. Data and graph were presented as medians (interquartile range) and statistical analysis was performed with Mann�CWhitney��s U-test. A value of P?