The width and length of the microscopic area were measured using a calibration slide and the surface area on the microscope slide was calculated in the following way: surface area= width (180μ) × length (130μ) = 23, 400μ Then, the whole area was selected and measured on the screen and was 27549835264μ.
In mirror spatial position with vasodilatation, more dense vascular chromophilic microscopic areas are found, signifying more active metabolism in relation with more proliferative status.
To quantify the fibrosis area, microscopic areas were selected randomly at an original magnification of 100X.
According to the results obtained from morphometry of the peritumoral infiltrate, the average percentage of inflamed area per microscopic field was greater in the undifferentiated tumors (Group 3) when compared to the averages of the moderately differentiated tumor samples (Group 2), followed by the well-differentiated tumor samples (Group 1).
The fibrotic area was measured by microphotometry using slices stained with Masson's trichrome, and a fibrotic area ≥5% of the microscopic field was defined as fibrosis.
Selection and isolation of the candidate cells and/or microscopic areas to be captured was performed under direct microscopic visualisation, by using the Pix Cell II Laser Capture Microscope (Arcturus Engineering, Mountain View, CA, USA).
Based on the analysis of each sample, simple arithmetic means the inflamed area per microscopic field were obtained and the results were expressed in percentage of inflamed peritumoral area per group.
HCT116 cells seeded on coverslips in 12-well format were stained according to the manual of the ApopTag® Fluorescein In Situ Apoptosis Detection Kit (Merck Millipore), and the apoptotic area was quantified by microscopic analysis with an Olympus BX41 microscope and CellSense imaging software.
A representative area was selected for qualitative light microscopic analysis of the inflammatory cellular response with a 10× objective [ 18].
The number of cells in the scratched area was determined by manual counting of the spots in the microscopic images.
