Mucin secretion and membrane water channel expression in the colon. (A) Mucin secreted from crypt layer cells was stained with Alcian blue at pH 2.5, and images were observed at 400× magnification. Five-to-six mice per group were used in preparation of Alcian blue stained colon tissue. (B) The levels of MUC2 and AQP8 transcripts in the total mRNA of colons were measured by semiquantitative RT-PCR using specific primers. After the intensity of each band was determined using an imaging densitometer, the relative levels of MUC2 and AQP8 mRNA were calculated based on the intensity of actin as an endogenous control. Five to six mice per group were used in preparation of total RNA and specific transcript level was assayed in duplicate in each test. (C) Quantitative real-time PCR analyses were performed to evaluate the levels of MUC2 and AQP8 transcripts in the similar way described as described in Figure 3B. Data are reported as the mean ± SD. *, p < 0.05 compared to the untreated group. #, p < 0.05 compared to the Lop + Vehicle treated group. No, Untreated group; Lop, Loperamide; SpiA, Spicatoside A, RT-PCR; Reverse transcription-polymerase chain reaction, MUC2; mucin 2, AQP8; aquaporin 8.