Quantitative Immunohistochemical Analysis of IL-33 and Proliferation Markers

Tissue sections were incubated with primary antibodies for anti-human IL-33 (R&D Systems, Minneapolis, MN, USA), anti-mouse Ki67, CD31, SA1009, vascular endothelial growth factor (VEGF; all obtained from Abcam, Cambridge, UK), or matching IgG isotypes overnight. Then, the sections were conjugated with secondary antibody. For the tissue array, Pannoramic MIDI (3DHISTECH, Budapest, Hungary) was used to scan the tissue points. All the immunohistochemical reactions were evaluated by three experienced pathologists who were blinded to clinical information according to the H-score method [26 (link)]. The H-score was calculated as follows: $$\begin{array}{cc}\hfill \text{H-score}& =\left(\text{percentage of cells of weak intensity},\times ,1\right)+\left(\text{percentage of cells of moderate intensity}\times 2\right)\hfill \\ \hfill & +\left(\text{percentage of cells of stringe intensity}\times 3\right)\hfill \\ \hfill \end{array}$$
The maximum H-score was defined as 300, based on 100% cells exhibiting strong intensity. The experiment was independently repeated three times.