Mouse anti brdu

Pancreatic islets were isolated from 8 to 10-week-old male C57BL/6 mice as previously described (Jun et al., 1999 (link)). Intact islets were dissociated at 37°C in Accutase (Millipore), given STZ (1 mM) for 15 h, washed with fresh media, and then cultured with BTC (1 nM). The islet cells or the αTC1-9 cells were fixed in 4% paraformaldehyde, permeabilized in permeabilization buffer (Thermo Fisher Scientific), blocked in blocking solution (Thermo Fisher Scientific), and then incubated with mouse anti-glucagon (Sigma, 1:100), rabbit anti-glucagon (DAKO, 1:100), mouse anti-PDX-1 (DSHB, Iowa, IA, 1:100) or mouse anti-BrdU (DAKO, 1:50) antibodies. FITC-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, 1:200) or TR-conjugated goat anti-mouse IgG or anti-rabbit IgG (Santa Cruz Biotechnology, 1:200) were used as secondary antibodies. Fluorescence was imaged using a laser scanning confocal fluorescent microscope (LSM 700).

The paraffin sections were stained as previously described [12] (link). Antibodies used were guinea pig anti-swine insulin (Dako Cytomation, Glostrup, Denmark), mouse anti-BrdU (Dako Cytomation), rabbit anti-survivin (Cell Signalling Technology), rabbit anti-C-peptide (Cell Signalling Technology, Danvers, MA, USA) and rabbit anti-ChromograninA (Dako Cytomation). Specificity of the survivin antiserum was confirmed by preabsorbtion with a survivin blocking peptide for 30 min (Cell Signalling Technology).
For light microscopy stainings the sections were incubated for 30 min at room temperature with a biotinylated secondary antibody (Zymed Laboratories, South San Francisco, CA, USA), rinsed and incubated with peroxidase-conjugated streptavidin (Zymed Laboratories). The sections were finally developed with 3-amino-9-ethyl-carbazole substrate (Thermo Scientific). For fluorescence microscopy, the secondary antibodies used were goat anti-guinea pig IgG (Alexa, A11076, Invitrogen, Paisley, UK), donkey anti-rabbit IgG (Alexa, A21206, Invitrogen) and donkey anti-mouse IgG (Alexa, A21202, Invitrogen). Nuclear staining was performed with DAPI (Vectashield with DAPI, Vector Laboratories, Burlingame, CA, USA).

Following 23 h treatment, the cells were further incubated with 1 µM BrdU (5′-bromodeoxyuridine, Sigma, St. Louis, Missouri, USA) for 1 h. The cells were then fixed with 4% PFA and cytospins were prepared. Following a denaturation step of 20 min with 2 M HCl, the cells were incubated with mouse anti-BrdU (1:10, Dako) and further incubated with fluorescein-labeled rabbit anti-mouse antibody (1:100, Dako), as previously described [68 (link),69 (link)]. Slides were mounted in Vectashield Mounting Media with DAPI (Vector Laboratories Inc., Burlingame, CA, USA) and the cells were observed in a DM2000 microscope (LEICA, Wetzlar, Germany). A minimum of 500 cells were counted per slide.

The following studies were approved by the Duke University Institutional Animal Care and Use Committee (IACUC). We delivered 30 mg/kg of GNF-9228, GNF-1346, or GNF-4088 suspended in DMSO by intraperitoneal injection into normal C57BL6 mice and measured levels of the compounds in the blood by mass spectrometry over the following 24 hours. In a separate study, 6 C57BL6 mice received daily injections of 30 mg/kg GNF-9228 for one week, while 6 control mice received injections of DMSO. For all mice in this second study, BrdU was added to the drinking water at a concentration of 0.8 mg/ml. 24 h after the final GNF-9228 or DMSO injection, the mice were euthanized and pancreata were dissected, fixed in neutral-buffered formalin, and paraffin embedded. Slides were incubated overnight with guinea pig anti-insulin (Dako) and mouse anti-BrdU (Dako) antibodies, followed by detection with an AlexaFluor 488 conjugated goat anti-guinea pig and AlexaFluor 555 conjugated goat anti-mouse secondary antibody (Invitrogen), and counterstaining with DAPI. Images were captured and analyzed using OpenLab software. A minimum of 4 slides per pancreas spaced 75–100 μm apart were analyzed, comprising a total of approximately 10,000 cells per condition.

C57BL/6 mice were sacrificed at 4 weeks after rAd-BTC or rAd-βgal injection. Pancreata were removed, fixed in 10% formalin, and embedded in paraffin. More than 200 serial sections (4 μm thick) were prepared from each pancreas, and every 20–25th section was used for immunohistochemical analysis. The tissue sections were boiled (100°C for 10 min, 10 mM sodium citrate, pH 6.0) for antigen retrieval, and blocked with blocking solution (DAKO, Carpinteria, CA, USA). The sections were then incubated with primary antibody solution: guinea-pig anti-insulin (DAKO, 1:100), rabbit anti-glucagon (DAKO, 1:100), mouse anti-PDX-1 (DSHB, Iowa, IA, 1:100) or mouse anti-BrdU (DAKO, 1:50). Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, 1:200), Texas Red (TR)-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, 1:200) or Alexa-Fluor-633-conjugated goat anti-guinea-pig IgG (Thermo Fisher Scientific, Rockford, IL, 1:200) were used as secondary antibodies. Fluorescence was imaged using a laser scanning confocal fluorescent microscope (LSM 700, Carl Zeiss MicroImaging, Jena, Germany) and colocalization was analyzed using the ZEN 2009 Analysis Program.