Gastric mucosal cells were isolated essentially as previously described, without Percoll gradient treatment [57 (link)]. Cell suspensions were stained for surface markers specific to epithelial cells and macrophages using the following monoclonal antibodies: rat anti-mouse E-cadherin (FITC, 1:100, clone #114420, R&D system); CD45 (Alexa Fluor 700, clone 30-F11, 1:500), CD11b (BV421, clone M1/70, 1:500), I-A/I-E (PE/Cy7, clone M5/114.15.2, 1:100); mouse anti-mouse CD64 (PE, clone X54-5/7.1, 1:100), CX3CR1 (APC, clone SA011F11, 1:200) (all from BioLegend). Dead cells were excluded by propidium iodide dye staining. Cells were sorted by flow cytometry into epithelial cells (E-cad+), macrophage enriched and depleted subsets using gating strategy as summarized (Figure 4D ). Total RNA was extracted from each population using RNeasy Mini Kit (Qiagen), DNase treated, converted to cDNA and analyzed by qRT-PCR as described above.
Rat anti mouse e cadherin
Manufactured by R&D Systems
The Rat anti-mouse E-cadherin is a laboratory reagent used for the detection and identification of E-cadherin, a cell-cell adhesion molecule, in mouse samples. It is a primary antibody raised in rat that specifically binds to the mouse E-cadherin protein.
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2 protocols using rat anti mouse e cadherin
1
Flow Cytometric Isolation and Analysis of Gastric Mucosal Cell Subsets
2
Western Blot Analysis of Cellular Proteins
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Cells were lysed by treating with CelLytic™ MT Cell Lysis Reagent (Sigma-Aldrich). An NE-PER™ kit (Pierce Biotechnology, Inc., Rockford, IL, USA) was used to isolate nuclear and cytoplasmic proteins. An equimolar quantity of protein was then subjected to SDS-PAGE, following which proteins were transferred onto nitrocellulose filters. The blots were blocked with 2% bovine serum albumin solution for 1 h and subsequently incubated overnight at 4°C with polyclonal goat anti-mouse IL-6 (cat. no. sc-1265; Santa Cruz Biotechnology, Inc.), rabbit anti-mouse IL-6 receptor (IL-6R; cat. no. sc-660; Santa Cruz Biotechnology, Inc.), mouse anti-human VEGF (cat. no. MAB293; R&D Systems, Inc.), rat anti-mouse E-cadherin (cat. no. 748-EC; R&D Systems, Inc.), anti-human mouse STAT3 (cat. no. MAB1799; R&D Systems, Inc.) and polyclonal goat anti-human MMP-9 (cat. no. AF911; R&D Systems, Inc.) (1:1,200 dilution) antibodies. The membranes were further incubated with horseradish peroxidase-conjugated secondary rabbit anti-goat (cat. no. ab6741), rabbit anti-mouse (cat. no. ab6728) or goat anti-rabbit (cat. no. ab6721) IgG antibodies (dilution, 1:1,000–1:2,000; Abcam, Cambridge, MA, USA), for 1 h at room temperature. The film was developed using an X-ray processor and visually examined to determine the intensity of the protein band.
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