Cella software version 2

For hematoxylin and eosin (HE) staining, Sirius Red/Fast Green staining and immunohistological analysis, formalin-fixed and paraffin-embedded tissue blocks were sectioned at a standard thickness of 5 µm, deparaffinized with xylene, and stained as described previously [22 (link)] by applying the following antibodies: rabbit monoclonal anti-α-SMA (Abcam, Cambridge, United Kingdom; ab32575, 1:300) and rabbit polyclonal anti-CD3 (Sigma-Aldrich, St. Louis, MO, USA; C7930, 1:1000). Sirius Red/Fast Green staining was performed as described previously [23 (link)]. Microscopical images were taken with an Olympus^TM CKX41 microscope with the ALTRA 20 Soft Imaging System^TM and Cell^A software version 2.6 (Olympus Soft Imaging Solutions GmbH, Münster, Germany). IrfanView^TM software version 4.36 (Irfan Skiljan, Jajce, Bosnia) was used in order to process images.

Results are expressed as mean ± SEM. Comparison between groups was made using ordinary one-way ANOVA. A p-value < 0.05 was considered statistically significant. Contingency table analysis and two-sided Fisher's exact tests were used to study the statistical association between categorical clinicopathological and immunohistochemical variables. Retrospective overall survival and progression-free survival curves comparing patients with and without any of the variables were calculated using the Kaplan-Meier method, with significance evaluated using log-rank statistics. For the progression-free survival analysis, patients were censored at the time of their last tumor-free clinical follow-up appointment. For the overall survival analysis, patients were censored at the time of their last clinical follow-up appointment. Median overall survival time for censored patients was 63.5 months (range: 3-135 months). Calculations were performed by using the GraphPad Prism Software version 6.01 (GraphPad Software, San Diego, CA, USA) and SPSS Software version 21.0 (SPSS, Chicago, IL, USA).
Microscopical images were taken using an Olympus™ CKX41 microscope with the ALTRA 20 Soft Imaging System™ and Cell^A software version 2.6 (Olympus Soft Imaging Solutions GmbH, Münster, Germany). Images were processed using IrfanView™ software version 4.36 (Irfan Skiljan, Jajce, Bosnia).

Values are presented as mean ± SEM or as mean ± SD in case of in vivo experiments. Comparison between groups was made using the Student's unpaired t-test. A p value <0.05 was considered statistically significant. All calculations were performed using the statistical computer package GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, USA).
Microscopical images were taken using an Olympus™ CKX41 microscope with the ALTRA 20 Soft Imaging System™ and Cell^A software version 2.6 (Olympus Soft Imaging Solutions GmbH, Münster, Germany). Images were processed using IrfanView™ software version 4.36 (Irfan Skiljan, Jajce, Bosnia).