P0141

Primary antibodies used include mouse anti-p120-catenin (1:500, BD Biosciences 610134), mouse anti-E-cadherin (1:200, BD Biosciences 610182) rat anti-CK8 (1:125, Developmental Studies Hybridoma Bank, Troma-1), rabbit anti-CK14 (1:10.000, Covance, PRB-155P), guinea pig anti-vimentin (1:400, Fitzgerald, 20R-VP004), rabbit anti-δ-catenin (EMD-millipore, 07–259), guinea pig anti-ARVCF (1:100, previously used in [33 ]), guinea pig anti-p0071 (1:100, previously used in [33 ]).
Secondary antibodies used were rabbit anti-guinea pig (DAKO, p0141), HRP conjugated rabbit anti-rat (DAKO p0450), poly HRP anti-rabbit (Immunologic, DPVR500HRP), poly HRP anti-mouse/rabbit/rat (Immunologic, DPVO500HRP), Alexa568 conjugated anti-mouse (1:500, Invitrogen, A11031) and Alexa488 conjugated anti-rabbit (1:500, Invitrogen, A11034).

Anti-XRCC4 rabbit polyclonal antibody [47 (link)], anti-FLAG mouse monoclonal antibody (clone M2; F3165; Sigma-Aldrich; St. Louis, MO, USA), anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) mouse monoclonal antibody (clone 6C5; MAB374;) and anti-LIG4 guinea pig polyclonal antibody (gifted by Prof. Miki Shinohara, Kinki University) [36 (link)] were used as the primary antibody at 1/1000 to 1/5000 dilution. As the secondary antibody, horseradish peroxidase-conjugated anti-rabbit immunoglobulins swine polyclonal antibody (P0399; Dako; Glostrup, Denmark), anti-mouse immunoglobulins goat polyclonal antibody (P0447; Dako) or anti-guinea pig immunoglobulins rabbit polyclonal antibody (P0141; Dako) was used at 1/1000 to 1/3000 dilution. The immunocomplexes were developed using WesternSure Chemiluminescent Western Blot Reagent (LI-COR; Lincoln, NE, USA) and the images were captured by C-Digit Blot Scanner (LI-COR). Protein Ladder One Plus, Triple-color (Nacalai Tesque) was used as the molecular weight standard. Other procedures of western blotting followed earlier publications [31 , 32 (link)].

After 31 weeks of study, o/n fasting animals were sacrificed by decapitation. The pancreas and fat tissues (retroperitoneal, epidydimal, mesenteric, subcutaneous, and brown fat) were immediately removed, weighted, and fixed in 10% buffered formalin.
Fixed pancreas samples were embedded in paraffin blocks, cut at a thickness of 3 µm and analyzed by immunohistochemistry (n = 4 for each group). Immunolabelling was performed with an antibody against insulin (dilution 1:8000, A0564 Dako) and a secondary antibody labeled with HRP (dilution 1:100, P0141 Dako). All sections were observed under an optical microscope using the 10× objective lens (Olympus CH, Shinjuku, Japan) and insulin positive cells were counted. Nine serial sections were analyzed for each pancreas.
The total area (mm²) of the analyzed sections was calculated. For this purpose, slides containing the stained histological samples were digitized (APERIO CS2, Leica Biosystems, San Diego, CA, USA) and images were analyzed using the ImageJ 1.52 software. The results of the quantification are shown as insulin-positive cells per pancreas area (insulin positive cells/mm²) at the end of the study.