Formalin-fixed tissues embedded in paraffin were mounted and de-paraffinized according to standard laboratory procedure. The number of individuals in whom paraffin tissues were available; au-CC: 20, rb-CC: 18, HC: 16, ref-CC: 14. Consecutive slides were stained with anti-CD3 (clone LN10, 1/200 dilution) (Leica Biosystems, Wetzlar, Germany) while haematoxylin was used as a counterstain. The Nikon E800 microscope (Nikon instruments Inc. Tokyo, Japan), equipped with a x 40 objective lens, and connected to the software NIS elements (Nikon instruments Inc. Tokyo, Japan) was used to acquire digital photos. The ImageJ programme (https://imagej.nih.gov/) was used in order to manually quantify the numbers of CD3+ lymphocytes in a minimum of eight unique fields of vision (Supplementary Image 1) in three consecutive slides. After un-blinding the data a median value of CD3+ cells was calculated for each individual. By using the flow cytometry percentages and the median value of CD3+ IHC cells we were then able to calculate the absolute number of CD3+ cells in each individual and the median values for each group (au-CC, HC, ref-CC, rb-CC) (34 (link)).
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