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. 2021 Apr 22;70(8):1538–1549. doi: 10.1136/gutjnl-2020-323868

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© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

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Immunohistochemistry for selected haemorrhoidal disease (HEM) candidate proteins. Illustration of the rectum and anal canal (A) indicating the site-specific localisation of the immunohistochemical panels analysed in (B). Results of fluorescence immunohistochemistry are shown for selected HEM candidate proteins encoded by HEM genes COL5A2 (rs16831319), SRPX (rs35318931), ANO1 (rs2186797), MYH11 (rs6498573) and ELN (rs11770437) (see also online supplemental table 11 and online supplemental figure 13). Antibody staining was performed on FFPE colorectal tissue specimens from control individuals. Picture layers correspond to the rectal mucosa (top row, epithelial surface delimited by a white dotted line, *=intestinal lumen), smooth musculature (second row), enteric ganglia (third row, ganglionic boundaries delimited by a white dotted line), haemorrhoidal plexus (fourth row, endothelial surface delimited by a white dotted line, *=vascular lumen) and the anoderm (bottom row, surface of the anoderm delimited by a white dotted line). Blue: DAPI; green: α-SMA (anti-alpha smooth muscle actin antibody) for rows 2 and 4 (smooth musculature/haemorrhoidal plexus) and PGP9.5 (member of the ubiquitin hydrolase family of proteins, neuronal marker) for row 3 (enteric ganglia); red: antibody for the respective candidate protein. Arrows point to respective candidate-positive cells within the vascular wall. Arrowheads point to respective candidate-positive nucleated immune cells.