Immunohistological Analysis of Tissue Sections

Five-μm-thick ice sections were stained with hematoxylin and eosin staining or subjected to immunohistology with an anti-α smooth muscle actin (α-SMA) antibody (ab5694, Abcam Japan, Tokyo, Japan), an anti-neutrophil antibody (ab2557, Abcam Japan, Tokyo, Japan), and an anti-Mac-3 antibody (550292, BD Pharmingen, Tokyo, Japan). To detect primary antibodies, sections for the anti-α-SMA antibody were incubated with the Dako Envision+ system HRP-labeled polymer anti-rabbit (ready to use) (Dako North America, California, USA) and sections for the anti-neutrophil and anti-Mac-3 antibodies were incubated with polyclonal rabbit anti-rat immunoglobulins/HRP (Dako North America, California, USA). These immunohistological staining was performed in accordance with our previous studies [21 (link),25 (link),30 (link),31 (link)]. Negative control slides were obtained by omitting each primary antibody.