Peyer’s patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. in this environment, we provide a protocol for preparing total mouse PP cells for flow cytometric and functional analysis, as well as a protocol for preparing PP cryosections for immunofluorescence microscopy and immunohistology. Our protocol for the isolation, characterization, and immunostaining of mouse PP cells is not novel articles. The first, by Fukuda and colleagues, describes the use of ligated ileal loop assays to assess the uptake of pathogenic bacteria by PP M cells 12. Mocetinostat The other, by Geem and colleagues, describes the isolation and characterization of DCs and macrophages from the mouse intestinal mucosa, but explicitly excludes PPs from their analysis 13. Protocol Animals were housed under conventional, specific pathogen-free conditions and were treated in full compliance with the Wadsworth Center’s Institutional Animal Care and Use Committee (IACUC) guidelines. 1. Oral Gavage (Optional) Gavage mouse strain of choice with antigen or microbes of interest using a 22 G Mocetinostat x1.5-in. blunt-end feeding needle (Popper Scientific, New Hyde Park, NY). Delivery volumes should not exceed 400 l per mouse. 2. Isolation of PP Cells for Flow Cytometry Euthanize mice by CO2 asphyxiation, according to institutional animal care and use committee (IACUC) guidelines. Cleanse the abdomen with 70% ethanol or betadine prior to surgery. Perform a standard laparotomy that involves a single (1 cm) incision using surgical grade scissors along the midline Mocetinostat beginning about 1.5 cm from the base of the rib cage. Expose the peritoneal cavity and identify the cecum. Snip the terminal small intestine at the ileal-cecal junction and gently remove the intestine in its entirety. Take care not to hyperextend the intestinal tissue as it is being removed from the peritoneal cavity. Lay the small intestine on a bed of moist paper towels or Kimwipes. Wet the small intestine gently with saline to prevent tissue dehydration. Visually identify individual PPs located on the anti-mesenteric side of the intestine (Figure 1). Typically, a single mouse has 5 to 10 visible PPs that are evenly distributed from the duodenum (proximal) to ileum (distal). On average, four mice will yield 23 PPs. Using curved surgical scissors, gently excise individual PPs and place them in cold Hank’s Balanced Salt Solution (HBSS). Note, the scissors should be placed curve-side Rabbit Polyclonal to Merlin (phospho-Ser518) up just above the PP and then gently applied to the tissue. Excise only the PP (not surrounding tissue) and transfer to cold HBSS. rat anti-mouse CD16/CD32, BD Biosciences), we simply use spent medium from a rat B cell hybridoma (ATCC 2.4.G2) that secretes a monoclonal IgG1 against murine Fc receptors for this step. Subject plate to gentle centrifugation (5 min at 1,000 x g) as above, and decant supernatant by gently inverting the plate. Add fluorophore-conjugated antibodies directly to cell suspensions at desired dilution, and incubate on ice for 30 min with continuous rocking. Subject plate to gentle centrifugation (5 min at 1,000 x g), and decant supernatant by gently inverting the plate. Clean cells with 100 d of stream stream. Centrifuge dish at 1,000 a g for 5 minutes, and decant supernatant by carefully inverting the dish. Resuspend cells 400 d fixation stream, which comprises of 300 d of stream stream and 100 l of 1% paraformaldehyde in PHEM buffer (60 mM Water lines, 25 mM HEPES, 10 mM EGTA, 4 mM MgCl2 at pH 6). Subject.