Computational protocol: Islet-1 Is Essential for Pancreatic β-Cell Function

Similar protocols

Protocol publication

[…] Pancreata were dissected, fixed in 4% paraformaldehyde (pH 7.0) for 6 h at 25°C, and embedded in paraffin or optimal-cutting-temperature compound (Tissue-Tek, 4583). Sections were blocked using CAS-Block (Invitrogen, 008120), and primary antibodies were applied overnight at 4°C. Primary and secondary antisera information is provided in Supplementary Tables 1 and 2, respectively. For immunofluorescence, Vectashield mounting medium with DAPI (Vector, H-1200) was used to counterstain nuclei, and fluorescein isothiocyanate tyramide signal amplification (PerkinElmer, NEL741001KT) was used for Isl-1 detection. For immunohistochemistry, signal was detected using Vectastain Elite ABC Kit (standard; Vector, PK-6100) and DAB Peroxidase Substrate Kit (Vector, SK-4100). Staining was visualized using a Leica DM6000 B microscope, and images were captured using the Leica LAS AF software and Leica DFC300 FX digital camera.To quantify staining, slides were digitally scanned using an Aperio ScanScope CS2 or MetaMorph microscopy automation software and analyzed using ImageScope software. Isl-1 ablation efficiency for a hormone+ population was calculated as the percentage of Isl-1+, hormone+ cells per total hormone+ cells using Indica Laboratories image analysis algorithms. β-Cell mass was calculated by averaging the percentage of insulin-stained tissue area over three sections that were taken at 100 μm levels. The fraction of positive area was then multiplied by the wet mass of the dissected pancreas measured at tissue harvest. Terminal deoxynucleotidyl TUNEL was performed as described () on three sections taken at 40 μm from pancreata that were harvested 14 days after the first Tm injection. These sections were then costained for insulin. TUNEL+, insulin+ cells were counted manually and normalized to the number of total β-cells. The number of total β-cells was determined by counting the Nkx6.1+ nuclei on an adjacent section. [...] Total RNA was extracted from pancreatic islets isolated by the standard collagenase P (Roche, 11 213 873 001) protocol () or whole pancreata. Total RNA preparation and cDNA synthesis were performed as described (). Quantitative PCR (qPCR) reactions were performed using SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich, S4438) and a Stratagene Mx3005P qPCR system. Fold enrichment of mRNA message was calculated by normalizing to a reference gene (see Supplementary Table 3 for qPCR primers). Control and mutant-isolated islet total RNA extractions were matched for pancreatic endocrine purity as described (). Microarray analysis was performed by the University of Pennsylvania’s Diabetes Research Center Functional Genomics Core. RNA was labeled with the Agilent Low Input Kit and hybridized, using a dye-switch design, to the Agilent 4 × 44K Whole Mouse Genome Microarray. Arrays were hybridized overnight and scanned using the Agilent Microarray Scanner. Data were normalized using normalizeBetweenArrays from the Limma package followed by SAMR to identify differentially expressed genes. […]

Pipeline specifications

Software tools limma, SAM
Application Gene expression microarray analysis
Organisms Mus musculus
Diseases Insulinoma
Chemicals Glucose, Tamoxifen