Ultraview dab

Immunohistochemistry (IHC) was performed using the 4B5 anti-HER2 primary antibody and a polymer-based detection system (UltraView DAB) on a BenchMark automated staining system (all by Ventana Medical Systems Inc., Tucson, USA). Peroxidase-conjugated secondary antibodies were used for chromogenic detecting by oxidizing 3,3′-Diaminobenzidin according to the manufactures protocol.
IHC HER2 stained slides were digitized using a Pannoramic P250 whole slide scanner (3D Histech, Budapest, Hungary) at 5.11 pixel/μm. DAB-precipitate thickness was measured with ‘ImageJ’ image-analysis software [18 (link)]. The regions of interest (ROIs) were manually defined according to the following rules: 10 non-adjacent tumor cells were measured per specimen. For each cell, ROIs perpendicular to the precipitate were drawn at 4 positions, i.e. 40 measurements per specimen, 4800 measurements in total. DAB-precipitate intensities were calculated using a color deconvolution algorithm [19 (link)]. The mean ROI-length and color intensity was calculated for each cell. The 8bit grey-scale intensity-values (0 = black, 256 = white) are as stated inverted, relative values to facilitate interpretation (0% = white, no staining; 100% black, full staining-intensity).

For immunohistochemical labelling, formalin-fixed paraffin-embedded aortic tissue sections of LDS patients or healthy donors were pre-treated in citrate buffer and incubated with antibodies against Gremlin-1 (bs-1475R, Bioss, Woburn, MA) in an automated stainer (Ventana Medical Systems, Tucson, AZ) according to a standard protocol (CC1st). For double labelling with Gremlin-1 and muscle actin (ENZ-30931, Enzo Life Sciences GmbH, Loerrach, Germany) or Gremlin-1 and CD34 (M7165, Dako, Hamburg, Germany), incubation with Gremlin-1 antibodies was followed by a short denaturing step and incubation with the second antibody. Bound antibodies were detected by the peroxidase method using diaminobenzidine as chromogen (760–500, Ultraview DAB, Ventana). For double labelling studies, bound Gremlin-1 antibodies were visualized with DAB as described above and expression of muscle actin or CD34 was demonstrated by alkaline phosphastase linked secondary antibodies using fast red as chromogen (760–501, Ultraview Universal Detection Kit, Ventana).

Immunohistochemistry (IHC) was performed in cut sections of pancreatic tumors using anti-MUC1 monoclonal antibody (MAb) clone 014E (MAb MUC1/014E, the kind gift of Suguru Yonezawa) [38 (link)] and anti-MUC4 MAb clone 8G7 (MAb MUC4/8G7, the kind gift of Surinder K. Batra) [39 (link)] using the immunoperoxidase method. Antigen retrieval was performed using CC1 antigen retrieval buffer (pH 8.5, EDTA, 100°C, 30 minutes; Ventana Medical Systems, AZ, USA) for all sections. Following incubation with the primary antibodies (MAb MUC1/014E diluted 1:5, 37°C, 32 minutes; MAb MUC4/8G7 diluted 1:3000, 37°C, 32 minutes) in phosphate buffered saline, pH 7.4 (PBS) with 1% bovine serum albumin (BSA), sections were stained on a Benchmark XT automated slide stainer using a diaminobenzidine detection kit (UltraView DAB, Ventana Medical Systems). The control staining (normal mouse serum or PBS-BSA instead of the primary antibodies) showed no reaction.

Immunohistochemistry (IHC) was performed in cut sections of lung tumors using anti-MUC4 MAb clone 8G7 (MAb MUC4/8G7, the kind gift of Surinder K. Batra) [9 (link)] using the immunoperoxidase method. Antigen retrieval was performed using CC1 antigen retrieval buffer (pH 8.5, EDTA, 100°C, 30 minutes; Ventana Medical Systems, AZ, USA) for all sections. Following incubation in phosphate buffered saline, pH 7.4 (PBS) with 1% bovine serum albumin (BSA), sections were stained on a Benchmark XT automated slide stainer using a diaminobenzidine detection kit (UltraView DAB, Ventana Medical Systems). The control staining (normal mouse serum or PBS-BSA instead of the primary antibodies) showed no reaction.