Immunohistochemical Detection of Somatostatin sst2a Receptors in the Lymphatic, Smooth Muscular, and Peripheral Nervous Systems of the Human Gastrointestinal Tract: Facts and Artifacts
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-spe- cific antibody R2- 88 for immunohistochemical staining of cryostat and formalin-fixed, paraffin-embedded tissue sections. Germinal cen- ters of intestinal lymphatic follicles were immunostained, exhibiting a predominantly plasma membrane localization of the receptor. Sim- ilarly, nerve fibers and cells in the submucosal and myenteric plexus were stained for sst2A. Antibody preabsorption with 100 nmol/L an- tigen peptide abolished staining in all of these tissues, and immu- nohistochemical staining correlated with the labeling observed after receptor autoradiography using the sst2-preferring radioligand 125I- (Tyr3)octreotide. Cytoplasmic immunostaining was detected in gas- trointestinal smooth muscle cells and was inhibited by antibody pre- absorption 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 se- quence chosen for antibody production was unique. Moreover, stan- dard sequence alignment protocols were unable to identify the se- quences 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 dem- onstrates for the first time the presence of specific sst2A receptor protein by immunohistochemistry in the human gastrointestinal lym- phatic and nervous components, but not in gastrointestinal circular and longitudinal smooth muscle. (J Clin Endocrinol Metab 84: 2942- 2950, 1999)