Anti glucagon

Proteins extracted from cells and animal tissues were separated by SDS–Polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted on to PVDF membrane (Millipore, IPVH00010) following standard protocols. Primary antibodies used in this study were anti-Insulin (Santa Cruz Biotechnology USA; sc- 25840), anti-Glucagon (Santa Cruz biotechnology USA; sc-74825), anti-G6PASE-α (Santa Cruz Biotechnology USA; sc- 25840), anti-PEPCK-C (Santa Cruz biotechnology USA; sc-74825), anti-AKT (Santa Cruz Biotechnology USA; sc-8312), anti-CASPASE 3 (Pierce USA; PA5–16335) and anti-GAPDH (Santa Cruz Biotechnology USA; sc-25778) and secondary antibody used was HRP conjugated anti-rabbit IgG (Santa cruz Biotechnology USA; sc-2030). Chemiluminescence detection was performed with the chemiluminescence detection kit (Amersham^TM Western Blotting Detection Reagents, UK) according to the manufacturer’s instructions. The protein bands in western blots were quantified using Image J software.

The SDF-1α, Arx, Pax4, Insulin and Glucagon expression were assessed by western blotting analysis and samples were normalized to GAPDH. Protein extraction was blocked with PBS-5% fat-free dried milk at room temperature for 1 h and incubated at 4°C overnight with anti-SDF-1α (1:1000, Santa cruz), anti-Arx (1:1000, Santa cruz), anti-Pax4 (1:1000, Santa Cruz), anti-Insulin (1:1000, Santa cruz), anti-Glucagon (1:1000, Santa cruz), anti-Aldh1a3 (1:1000, Novus), anti-Neurog3 (1:1000, Beta Cell Biology Consortium), anti-MafA (1:1000, Cell Signaling Technology), anti-Pdx1 (1:1000, Cell Signaling Technology), anti-NeuroD1 (1:1000, Cell Signaling Technology) and anti-XBP1 (1:1000, Santa cruz), anti-CD63 (1:2000, Abcam), anti-TSG101 (1:1000, Santa Cruz), anti-Ago2 (1: 1000, Santa Cruz) and anti-GAPDH (1:3000, Santa Cruz) antibodies respectively.

Pancreatic and liver tissues were fixed with 10% formaldehyde and paraffin embedded by standard methods. Subsequently, the tissues were sectioned (5 µm thin) and stained with Hematoxylin & Eosin (H & E) and were mounted by mounting medium. At least 5 islets in each section were randomly chosen and analyzed. The cell number of each islet was quantified with Image J software (https://imagej.nih.gov/ij/). Tissue morphology was examined and analysed by clinical pathologist.
Pancreatic tissue sections were subjected to immunohistochemistry for insulin and glucagon using anti-Insulin (Santa Cruz Biotechnology USA; sc- 25840), anti-glucagon (Santa Cruz biotechnology USA; sc-74825) and anti-Ki-67 (Santa Cruz biotechnology USA; sc-101861) antibodies and HRP conjugated secondary antibody (Santa cruz Biotechnology USA; sc-2030) as per standard protocols. The sections were counter stained with Harris Hematoxylin (Nice Co., India) and the detection was based on the intensity of brown chromogen produced by HRP-DAB at the site of enzyme activity (3, 3′-diaminobenzidine) and the images were captured using light microscope. Subsequently for fluroscence imaging, anti-rabbit IgG-FITC (Sigma-Aldrich Inc., USA; F6005) and anti-mouse IgG-TRITC (Sigma-Aldrich Inc., USA; T5393) were used.

Mouse pancreatic tissues were collected, fixed in paraformaldehyde, dehydrated and then embedded in paraffin. Five‐micrometer thick sections were prepared for immunofluorescence staining. Briefly, after deparaffinization and rehydration, sections were blocked in 5% bovine serum albumin, followed by incubation with primary antibodies overnight at 4°C, and then with FITC‐ or TRITC‐conjugated secondary antibodies (Santa Cruz) for 60 minutes at 37°C. The sections were washed with phosphate‐buffered saline–Tween 20 three times, then mounted with DAPI (Abcam) for nuclear staining. For immunohistochemical staining, horseradish peroxidase‐conjugated anti‐rabbit secondary antibodies were used, and the staining was visualized using diaminobenzidine, followed by counterstaining with haematoxylin. The following primary antibodies were used: anti‐amylase (sc‐46657; Santa Cruz), anti‐insulin (sc‐9168; Santa Cruz), anti‐insulin (ab6995; Abcam), anti‐glucagon (sc‐13091; Santa Cruz), anti‐FGF21 (ab64857; Abcam), anti‐syntaxin‐1 (STX‐1) (sc‐12736; Santa Cruz), anti‐SNAP25 (ab41455; Abcam), anti‐VAMP2 (ab3347; Abcam) and anti‐cleaved caspase‐3 (9664; Cell Signaling Technology).

After 6 weeks of experiment, the whole pancreatic tissue was resected from each mouse and weighed. These were fixed in 10% neutral buffered formalin, blocked in paraffin, and sectioned at 4 µm on an automatic rotary microtome (Zeiss, Jena, Germany). For immunofluorescence, the sections of the pancreatic portions were stained overnight with the target antibodies: anti-insulin (#sc-9168, Santa Cruz biotechnology, Dallas, TX, USA) and anti-glucagon (#SC-7779, Santa Cruz biotechnology, Dallas, TX, USA). Tissues were washed with phosphate buffered saline (PBS) and incubated with the secondary antibodies: anti-rabbit Alexa 594 (Life Technologies, Carlsbad, CA, USA) and anti-goat Alexa 488 (Life Technologies, Carlsbad, CA, USA). In the case of PCNA staining, tissues were incubated with the primary PCNA antibody (Vector lab, Burlingame, CA, USA) overnight. After washing with PBS, the sections were peroxidase-labeled using the Vector-statin ABC system (Vector lab, Burlingame, CA, USA), then developed with 3,3′-diaminobenzidine (DAB), and counterstained with Meyer’s hematoxylin. A BrdU assay was performed in accordance with manufacturer’s protocol (Sigma, USA). RNA in situ hybridization was performed as previously described [33 (link)].

Antibodies to GLUT4 (no. sc-7938) were purchased from Santa Cruz Biotech (Santa Cruz, Paso Robles, CA, USA); phospho-AMPK (Thr¹⁷²), PPARα (no. ab8934), and PPARγ (no. ab45036) were obtained from Abcam Inc. (Cambridge, MA, USA); FAS (no. 3180), phospho-Akt (Ser⁴⁷³) (no. 4060), total AMPK (Thr¹⁷²), phospho-FoxO1 (Ser²⁵⁶) (no. 11115), total-FoxO1 (Ser²⁵⁶) (no. 2880), and β-actin (no. 4970) were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary anti-rabbit antibodies were obtained from Jackson ImmunoRes. Lab., Inc. (West Grove, PA, West Baltimore Pike, USA). Anti-insulin (1 :100, no. sc-9168) or anti-glucagon (1 : 200, no. sc-13091) primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz Inc., Paso Robles, CA, USA).

Immunohistochemistry procedures have been described previously [18 (link)]. Anti-insulin (1:200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, #sc9168), anti-glucagon (1:200, Santa Cruz Biotechnology, #sc13091), or anti-somatostatin (1:200, Abcam, #ab15365) primary antibodies were used for pancreas immunostaining. Staining was developed using Histostain-Plus Broad Spectrum (AEC) Kit (Invitrogen, Frederick, MD). Slides were counterstained with hematoxylin to identify cell nuclei.
After staining, slides were rinsed in deionized water and placed on coverslips in Clear Mount mounting medium (Electron Microscopy Sciences, Hatfield, PA). The specificity of immunoreactivity was confirmed by omitting the primary antibody from some sections. The staining was observed using a light microscope Nikon Eclipse 80i (Nikon Instruments, Melville, NY). Images were analyzed using Adobe Photoshop CZ4 extended software.