Mouse Spi-B Antibody

Spi-B in Mouse Splenocytes. Spi-B was detected in immersion fixed mouse splenocytes using Sheep Anti-Mouse Spi-B Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7204) at 15 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (red; Catalog # NL010) and counterstained with DAPI (blue). Specific staining was localized to plasma membranes and cytoplasm. View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

Detection of SPi-B in Mouse Splenocytes by Flow Cytometry. Mouse splenocytes were stained with Sheep Anti-Mouse Spi-B Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7204) followed by Allophycocyanin-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # F0127) and Rat Anti-Mouse B220/CD45R PE-conjugated Monoclonal Antibody (Catalog # FAB1217P). Quadrant markers were set based on control antibody staining (Catalog # 5-001-A). To facilitate intracellular staining, cells were fixed with paraformadehyde and permeabilized with saponin.

Detection of Mouse Spi-B by Immunocytochemistry/Immunofluorescence OPGhigh M cells cluster more in cecal patches than in Peyer’s patches.a Whole-mount immunostaining of the FAE of Peyer’s patches (left) and cecal patches (right) for OPG (green) and Spi-B (red). Nuclei were stained with DAPI (blue); scale bars: 50 µm. b Scatter plots of the fluorescence intensities of OPG versus Spi-B. Red dots represent cells stained with anti-Spi-B antibody that was conjugated with HyLyte Fluor 555 and anti-OPG antibody that was conjugated with HyLyte Fluor 647. Blue dots represent background fluorescence intensity of randomly selected non-stained cells. Fluorescence intensities were measured for at least 3000 cells from five FAEs of three mice. c Frequencies of OPGhigh M cells in Peyer’s patches and cecal patches were quantified. *p < 0.05, ***p < 0.005. Student’s t-test, n = 5 FAE from three animals. The source data underlying panels b and c are provided as a Source Data file. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31932605), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Spi-B by Immunocytochemistry/Immunofluorescence RANKL–RANK signaling is stimulated in the gut epithelia of Opg−/− mice.a GP2+ cells (red) are more effectively induced in the cecal epithelium (CE) and ileal villi (VE) of Opg−/− mice by RANKL administration. Whole-mount immunohistochemical images of Spi-B (green) and GP2 (red) in the VE and CE of WT (upper panels) and Opg−/− mice (lower panels) treated with either GST (control; left) or GST-RANKL (right). Scale bars: 100 µm. Representative images from three independent experiment are shown. b Quantitative PCR analysis of M-cell marker expression in conventional epithelia from the VE and CE of mice injected with GST (control) or GST-RANKL. Results were normalized to Gapdh expression and are presented relative to the expression in the ileal epithelium from GST-treated mice. Data shown are mean values from three independent experiments (error bars indicate standard deviation). ***p < 0.005, **p < 0.01, *p < 0.05; p values were calculated with the Student’s t-test (n = 3 biologically independent experiments). c, d Nuclear translocation activities of RelB and p52 following RANKL stimulation are enhanced in Opg−/− mice. c Western blot analysis of p100/p52 and RelB in the VE and CE of mice injected with GST or GST-RANKL. Rpt4, a subunit of the 26S proteasome, was used as an internal control for the cytoplasmic fraction. Lamin A/C (Lamin) was used as an internal control for the nuclear fraction. Data are representative of two independent experiments. d Right, single confocal planes of the cecal FAE from WT and Opg−/− mice. FAE monolayers were stained with anti-RelB (green) and anti-Spi-B (red) antibodies and with Hoechst 33342. Left, a bar graph summarizing the proportions of RelB-positive cells among total numbers of M cells (Spi-B-positive cells). ***p < 0.005; p values were calculated with the Student’s t-test (n = 4 biologically independent experiments). Scale bars: 20 µm. The source data underlying panels b and d and non-cropped scan images of western blotting (c) are provided as a Source Data file. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31932605), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Spi-B by Immunocytochemistry/Immunofluorescence RANKL–RANK signaling is stimulated in the gut epithelia of Opg−/− mice.a GP2+ cells (red) are more effectively induced in the cecal epithelium (CE) and ileal villi (VE) of Opg−/− mice by RANKL administration. Whole-mount immunohistochemical images of Spi-B (green) and GP2 (red) in the VE and CE of WT (upper panels) and Opg−/− mice (lower panels) treated with either GST (control; left) or GST-RANKL (right). Scale bars: 100 µm. Representative images from three independent experiment are shown. b Quantitative PCR analysis of M-cell marker expression in conventional epithelia from the VE and CE of mice injected with GST (control) or GST-RANKL. Results were normalized to Gapdh expression and are presented relative to the expression in the ileal epithelium from GST-treated mice. Data shown are mean values from three independent experiments (error bars indicate standard deviation). ***p < 0.005, **p < 0.01, *p < 0.05; p values were calculated with the Student’s t-test (n = 3 biologically independent experiments). c, d Nuclear translocation activities of RelB and p52 following RANKL stimulation are enhanced in Opg−/− mice. c Western blot analysis of p100/p52 and RelB in the VE and CE of mice injected with GST or GST-RANKL. Rpt4, a subunit of the 26S proteasome, was used as an internal control for the cytoplasmic fraction. Lamin A/C (Lamin) was used as an internal control for the nuclear fraction. Data are representative of two independent experiments. d Right, single confocal planes of the cecal FAE from WT and Opg−/− mice. FAE monolayers were stained with anti-RelB (green) and anti-Spi-B (red) antibodies and with Hoechst 33342. Left, a bar graph summarizing the proportions of RelB-positive cells among total numbers of M cells (Spi-B-positive cells). ***p < 0.005; p values were calculated with the Student’s t-test (n = 4 biologically independent experiments). Scale bars: 20 µm. The source data underlying panels b and d and non-cropped scan images of western blotting (c) are provided as a Source Data file. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31932605), licensed under a CC-BY license. Not internally tested by R&D Systems.