Hrp dab envision plus kit

We utilized 2 sets of tumor microarrays (TMA): one set contained 88 chemonaïve mesothelioma samples (created by the Pathology Core facility at University of California San Francisco, Mt. Zion with anonymous mesothelioma samples obtained from our collaboration with Drs. Bueno and Richards) and one set contained a different 88 mesotheliomas with matching normal tissue, created from clinical pathology blocks of Brigham and Women's Hospital patients. In addition to anonymous control cores, microarray blocks contained, per included patient, 3 cores of tumor and, if available, 3 cores of normal tissue obtained from selected areas of archival clinical blocks. Sections of TMAs (5 μm) were stained for 2 h at RT with antibodies for ASS1 (clone 2C10, 1:1000, Millipore, Billerica, MA, USA) and visualized with a HRP/DAB Envision plus Kit (#K4010, Dako, Carpinteria, CA). Known negative and positive controls were included. Hematoxylin was used as a counterstain. Slides were viewed by two independent scorers and scored semi-quantitatively (0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining) [18 (link)].

Tumor fragment spheroids used for the apoptosis studies were probed for expression of Bim, Akt, P-Akt, P-S6K, Ki67, GLUT-1 and IRS-1 protein levels by immunohistochemistry. Paraffin sections (5 μm) were stained for 2 h at RT with antibodies for Bim (1:200 #559685BD, Pharmingen, San Jose, CA), P-AKT^Ser473 and P-S6K^Thr389 (1:100 #4060, 1:100 #9234, Cell Signaling, Danver MA, USA), Ki67 (1:200, #ab15580, Abcam, Cambridge, MA, USA), GLUT-1 and IRS-1 (1:500 PA5-16793, 1:800 PA5-29667, Thermo Scientific, Waltham, MA, USA) and visualized with a HRP/DAB Envision plus Kit (#K4010, Dako, Carpinteria, CA). Known negative and positive controls were included. Hematoxylin was used as a counterstain. Slides were viewed by two independent scorers and scored semi-quantitatively (0 = no staining, 1 = weak staining in 0–25% cells, 2 = moderate staining in 0–50% of cells, 3 = strong staining in 50–100% of cells).