Quantitative analyses
Results of the histochemical analyses were quantified in each zone of
the SLIL and control tissues using the ImageJ software (version 1.53k,
National Institute of Health, Bethesda, MD), as previously described (V.
Carriel et al., 2014; Ortiz-Arrabal et al., 2021; Rodriguez-Pozo et al.,
2020; Ruiz-López et al., 2022). In each image, we randomly selected five
25 µm × 25 µm square areas, and we obtained a numeric value (expressed
as intensity units or I.U.) corresponding to the average pixel intensity
within each square using the square tool of the software. Results were
then subtracted from the blank values and averages and standard
deviations were calculated for each group of samples. A total of 30
measurements were obtained for each type of tissue (controls and SLIL
zones). Results corresponding to the analysis of cell phenotypes as
determined by CD73 and S100 immunohistochemistry were quantified by
determining the percentage of positive cells found at in each region of
the SLIL. In short, we counted the total number of cells and the number
of cells showing positive and negative signal within a 300 µm × 300 µm
area randomly selected on each region, and the percentage of positive
cells was calculated. The same method was used to quantify the number of
blood vessels found at each SLIL region in samples subjected to CD34
immunohistochemistry.