PMC:4135037 / 25836-27236 JSONTXT

{"project":"2_test","denotations":[{"id":"24531709-17283130-79171899","span":{"begin":664,"end":666},"obj":"17283130"},{"id":"24531709-17283130-79171899","span":{"begin":664,"end":666},"obj":"17283130"}],"text":"Histopathology, Immunofluorescence, and Immunohistochemistry\nTissues of organs were removed and fixed in 10% neutral buffered formalin, or snap frozen in liquid nitrogen. The gross sizes of pituitaries or pituitary masses were measured using an electronic ruler. After macroscopic evaluation during necropsy, tissues were embedded in paraffin, sectioned at 5 μm thickness and stained with hematoxylin and eosin (H\u0026E). Pituitary and pancreatic sections were examined and photographed using a Zeiss Axiovert microscope and Axiovision software. Additional sections were taken for immunofluorescence and immunohistochemical analyses, performed as previously described 45. To measure proliferating and mitotic cells, sections were blocked with normal goat serum in phosphate-buffered saline and incubated with a polyclonal antibody against Ki67 (NCL-Ki67 at a dilution of 1:1,000; Novocastra Laboratories) and with biotin-conjugated secondary antibody (Vector Laboratories). Other primary antibodies used were anti-insulin (Zymed) and anti-Cdk4 (Santa Cruz). Immunocomplexes were detected using the Vectastain ABC alkaline phosphatase kit according to the manufacturer's instructions (Vector Laboratories). For quantification of Ki67-positive islet cells, at least 1,000 cells in total were counted within each category of the following three: nonhyperplastic islets, hyperplastic islets and islet tumors."}