Computational protocol: Significant Role of Collagen XVII And Integrin β4 in Migration and Invasion of The Less Aggressive Squamous Cell Carcinoma Cells

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Protocol publication

[…] Sections of 3.5 μm cut from TMA specimens were deparaffinized and rehydrated. After antigen retrieval and neutralizing endogenous peroxidase activity, the sections were incubated with the polyclonal NC16A antibody (1:8000), the monoclonal laminin γ2 (sc-28330, 1:2000) and the polyclonal integrin β4 (sc-9090, 1:2000) (Santa Cruz Biotechnologies Inc., Santa Cruz, CA, USA). The EnVisionTM system (Dako Denmark A/S, Glostrup, Denmark) was used for detection. The histological images were captured with an Olympus DP25 camera (Olympus, Center Valley, PA, USA) attached to a Nikon Eclipse E600 microscope (Nikon, Tokyo, Japan) using x20 objective.Computer-assisted immunoreactivity evaluation was done using ImageJ (NIH, Bethesda, MD, USA), a freeware image analysis software, using a method described and validated earlier. Representative areas of the TMA punches were selected and the macro was used to calculate the proportion of positive immunoreaction in epithelial cells relative to total epithelial cell area in the selected region of interest representing normal skin, actinic keratosis, Bowen’s disease or SCC. TMA punches with insufficient tissue or tumor were excluded from the study. [...] The statistical analyses were conducted using IBM SPSS Statistics for Windows, version 22.0 (IBM Corp., Armonk, NY, USA). The independent sample t-test was used to analyze the statistical significances in immunoblotting, scratch wound healing and organotypic myoma assays. The statistical significances of the associations of the amount of positive immunoreaction in epithelial cells with different tumor types were analyzed by Kruskal-Wallis test (comparing three or more classes) or Mann-Whitney U test (comparing two classes). Spearman rank correlation coefficients (ρ) were used to study the correlation between different immunohistochemical markers. A two-tailed p value < 0.05 was considered statistically significant. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Human poliovirus 1 Mahoney