Publication protocol
Mice were sacrificed at an age of 20 wk by bleeding and cervical dislocation (12 per group, 6 male, 6 female). The pancreas was immediately removed, weighed and organ mass was related to whole body weight to obtain relative organ weights. Explanted mouse pancreata were fixed in formaldehyde solution (4%, OttoFischerGmbH, Saarbruecken, Germany) overnight, embedded in paraffin and cut into 5 μm thick longitudinal sections. One section was mounted on one slide. We analyzed six slides per pancreas (each tenth), which were equally distributed over the organ. Sections were dewaxed and rehydrated by xylol and a descending ethanol series. Subsequently, slides were boiled in target retrieval solution (pH 9, Dako, Hamburg, Germany) and cooled down for 15 min, followed by washing in PBST (Biochrom Berlin, Germany) thrice. Quenching of endogenous peroxidase-activity by incubation for 10 min with 3% H2O2 (Sigma-Aldrich) was performed twice, followed by another washing step. Sections were blocked with 5% donkey-serum (= host of secondary antibody, Sigma) for 30 min at room temperature (RT) and incubated with primary antibodies (AB) for insulin (C27C9) or glucagon (D16G10, both rabbit monoclonal AB, Cell Signaling, Cambridge, UK) for 60 min at RT. Slides were washed thrice and incubated with secondary AB (711-035-152, donkey anti-rabbit polyclonal AB, Dianova, Hamburg, Germany) for 30 min at RT. Staining was performed with DAB Peroxidase Substrate Kit (Liquid DAB+Substrate Chromogen System, Dako), following instructions of the manufacturer. Slides lacking the primary antibody were used as controls. Sections were counterstained with ready-to-use hematoxylin (Hollborn, Leipzig, Germany). Additional overview images were stained with hematoxylin (Hollborn) and eosin (0.2%, Medite, Burgdorf, Germany; HE). Stained sections were photographed with a Keyence Biozero BZ 8000 microscope. Staining intensity of DAB was quantified using a scoring system from negative to 6x positive. Quantifications of islets, area and beta cell mass (mg per pancreas) was calculated by multiplying relative DAB-positive area (the percentage of glucagon positive area over total pancreas area) by pancreas weight, as previously reported [30] using ImageJ software (ImageJ 1.45; http://rsbweb.nih.gov/ij/download.html).
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