Tumor and adjacent normal lung tissues were acquired at initial diagnosis of 13 NSCLC patients (ages 48–75; stage II–III; Ontario Tumor Bank), and subjected to IB analysis. Human lung cancer tissue microarrays (LC1005, LC1501, US Biomax, Inc.) were stained using the Discovery XT Staining System (Ventana Medical Systems, Inc.). Antigens were retrieved with an EDTA pH 8.0 solution and incubated with rabbit anti-CIP4 Ab #2 (1:10). CIP4 immunohistochemistry (IHC) staining was visualized using DAB and a hematoxylin counterstain. Microarrays were scanned using the Aperio CS digital slide scanner (Queen’s Laboratory for Molecular Pathology) and analyzed with ImageScope software (Aperio). Tumor-specific H-scores were calculated based on positive pixel intensity according to the formula: (% weak positive X 1) + (% positive X 2) + (% strong positive X 3). For analysis of CIP4 transcripts in lung adenocarcinoma microarray studies, a Kaplan-Meier curve for overall survival was created using Kaplan-Meier Plotter (www.kmplot.com ) with patients grouped according to high and low expression of Trip10. Hazard Ratio (with 95% confidence interval) and logrank P values were calculated and are displayed on the graph.
Discovery xt staining system
Manufactured by Roche
Sourced in United States
The Discovery XT Staining System is a laboratory instrument designed for the automated processing and staining of tissue samples. It performs various staining protocols to prepare samples for subsequent analysis. The system is capable of handling multiple slides simultaneously, providing a standardized and efficient staining process.
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3 protocols using discovery xt staining system
1
CIP4 Expression in NSCLC Tumors
2
Immunohistochemical Analysis of FBP17 Expression
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TMAs were stained using the Discovery XT Staining System (Ventana Medical Systems, Inc. Tucson, AZ, USA). IHC of the TMAs were done at the Department of Histopathology, PGIMER, Chandigarh. Antigens were retrieved with an EDTA pH 8.0 solution and incubated with FBP17 antibody at dilution 1:100. Staining was visualized with DAB treatment and a Hematoxylin counterstain. Analysis of the microarrays was done and Histological scoring (H Score) was given according to the percentage of cells stained (0 is <10%, 1+ is 10–25%, 2+ 25–50%, 3+ is 50–75%, 4+ above 75%) and intensity. The staining intensity of FBP17 was scored as weak, moderate and strong positive according to the brown intensity at the cytoplasmic portion. Similarly, we also analyzed the same tissue microarrays in terms of other parameters such as molecular subtypes, lymph node and grade of the tumor.
3
Immunohistochemical Analysis of p53 and Toca-1 in Breast Cancer Tissue Microarrays
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Human breast cancer tissue microarrays (T086b, BR10010a, BR963 and BR953, US Biomax, Inc., Rockville, MD, USA) were stained using the Discovery XT Staining System (Ventana Medical Systems, Inc. Tucson, AZ, USA). Antigens were retrieved with an EDTA pH 8.0 solution and incubated with rabbit anti-p53 (DO-1) and Toca-1 antibody as used previously [11 (link)],[23 (link)]. Staining was visualized with DAB treatment and a hematoxylin counterstain. Microarrays were scanned using the Aperio CS digital slide scanner (Queen’s Laboratory for Molecular Pathology) and analyzed with ImageScope software (Aperio). Tumor-specific H-scores were calculated based on positive pixel intensity according to the formula: (% weak positive X 1) + (% positive X 2) + (% strong positive X 3). The p53 IHC scoring was grouped according to the extreme positive (EP), extreme negative (EN), and non-extreme (NE) categories that were recently shown to best relate to p53 mutation status and outcomes [24 (link)]. Toca-1 H-scores were reported according to p53 EP/EN or NE groups, and analyzed by Student’s t test.
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