Figure 2. Preferential expression of Spi-B transcript in mouse M cells.

(a) The kinetics of Spi-B expression after RANKL-treatment was assessed by qPCR. Data represent fold change compared to the normalized value of expression of each transcript in epithelial cells from untreated mice. (b) Spi-B mRNA expression was measured by qPCR in villous epithelium (VE) and follicle-associated epithelium (FAE). Data represent fold change compared to the normalized value of expression of each transcript in villous epithelial cells. All samples were normalized to the expression level of GAPDH (n = 3). (c) In situ hybridization (ISH) analysis of Spi-B mRNA in the small intestine from RANKL-treated and untreated mice. Scale bar: 50 μm. (d) The top panel shows ISH image of Spi-B mRNA in a PP follicle. Scale bar: 100 μm. Lower three panels show the high magnification images of box in the top panel, after restaining of the section with UEA-I. Dotted lines represent FAE. Arrows indicate the double positive cells with Spi-B and UEA-I. Asterisks indicate goblet cells on adjacent villous epithelium, which are also positive for UEA-I. Scale bar: 20 μm. (e) Top panel shows immunostaining of Spi-B (red) in small intestine from mouse after 18 hours after RANKL-treatment. Lower three panels show dual staining of Spi-B (red) and GP2 (green). Arrows indicate Spi-B⁺GP2⁺ cells. All sections were counterstained with DAPI. Scale bar: 20 μm. (f) ISH of Spi-B mRNA in a PP from 18.5 day old wild-type mouse embryo is shown (top panel). Spi-B mRNA was also expressed in other GALT, such as ILFs, colonic patches and cecal patches (lower three panels). Scale bar: 50 μm. Data are representative of three independent experiments (error bars, s.d.).