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Immunohistochemical Detection of Somatostatin sst2a Receptors in the Lymphatic, Smooth Muscular, and Peripheral Nervous Systems of the Human Gastrointestinal Tract: Facts and Artifacts¹

Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne (J.C.R., J.A.L., B.W.), Berne, Switzerland; the Department of Cell Biology, Baylor College of Medicine and Biomedical Computing, Inc. (D.L.S.), Houston, Texas 77030; and the Department of Integrative Biology, Pharmacology, and Physiology, University of Texas Houston Medical School (R.W.H., A.S.), Houston, Texas 77030

Address all correspondence and requests for reprints to: Jean Claude Reubi, M.D., Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, P.O. Box 62, Murtenstrasse 31, CH-3010 Berne, Switzerland. E-mail: reubi{at}patho.unibe.ch

The cellular distribution of the somatostatin sst2A receptor protein was investigated in the lymphatic, smooth muscular, and nervous components of the human gastrointestinal tract using subtype-specific antibody R2–88 for immunohistochemical staining of cryostat and formalin-fixed, paraffin-embedded tissue sections. Germinal centers of intestinal lymphatic follicles were immunostained, exhibiting a predominantly plasma membrane localization of the receptor. Similarly, nerve fibers and cells in the submucosal and myenteric plexus were stained for sst2A. Antibody preabsorption with 100 nmol/L antigen peptide abolished staining in all of these tissues, and immunohistochemical staining correlated with the labeling observed after receptor autoradiography using the sst2-preferring radioligand ¹²⁵I-[Tyr³]octreotide. Cytoplasmic immunostaining was detected in gastrointestinal smooth muscle cells and was inhibited by antibody preabsorption with antigen peptide. However, ¹²⁵I-[Tyr³]octreotide autoradiography was negative, and Western blots showed no band at the usual 70–90 kDa location for sst2A. Instead, a band was observed at 205 kDa. This band comigrated with the rabbit myosin standard, which was also stained with R2–88, although antibody sensitivity for myosin was less than 0.002% of that for the sst2A receptor. Rigorous computer-based sequence analysis demonstrated the peptide sequence chosen for antibody production was unique. Moreover, standard sequence alignment protocols were unable to identify the sequences in myosin responsible for the observed reactivity with the R2–88 antiserum. The observed cross-reactivity emphasizes the need for extensive controls to prove the specificity of immunostaining for such low abundance proteins as receptors even when the peptide sequence chosen for antibody production is unique. This study demonstrates for the first time the presence of specific sst2A receptor protein by immunohistochemistry in the human gastrointestinal lymphatic and nervous components, but not in gastrointestinal circular and longitudinal smooth muscle.

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