C4 antibody recognizes small intestinal mucus preferentially by immunohistochemistry, Western blot, and ELISA. a) Immunohistochemistry. The C4 antibody (diluted 1:50) recognizes the epithelial lining of fetal mouse small intestine by immunohistochemistry. Note dark brown staining of specific cells. Magnification using 40× objective. b) Western blot hybridized with the C4 antibody. 23.4 μg of mucus collected from either the guinea pig small intestine (I) or stomach (S) was subjected to electrophoresis through a 1.0% agarose gel and then transferred to a nitrocellulose membrane. Hybridization with the C4 antibody (1:100) labeled a high molecular weight smear and a second high molecular weight band in the intestinal mucus, but did not recognize the stomach mucus. Western blot shown is representative of three separate blots. c)ELISA plate was coated with equal amounts of guinea pig stomach or small intestinal mucus (11.7 μg per well). The C4 antibody (1:500) preferentially recognizes the small intestinal mucus, although less differentiation is observed at lower working concentrations of the antibody. Each sample was assayed in triplicate, and data are shown as mean ± SEM. The assay was done twice using two independent sample isolations, with similar results obtained each time.