Mouse anti vimentin mab

A single dose of fluorescently labelled (WGA-Alexa Fluor 555; Thermo Fisher) living Lactobacillus Rhamnosus (LR) was given to naïve rats, and the gut tissue isolated after 30 minutes. Serial 10 μm thick cryosections of Peyer's patches were co-stained with mouse anti-vimentin mAb (Dako) and sections of villi were counterstained with mouse anti-cytokeratin mAb (Dako), followed by goat anti-mouse Ab Alexa Fluor 488 conjugated. Nuclei were stained with DAPI. Single z-scan images were captured via confocal microscopy (20x objective).

Total cell lysates were prepared for each of the EpCAM subpopulations, the four selected PMC42-LA clones, and and parental PMC42-LA cell line by lysing the cells in the presence of RIPA Buffer (10 mM Tris-HCl pH 7.6, 10 mM NaCl, 3mM MgCl2, 1% nonidet P-40, 1 X Protease Inhibitor tablet (Roche)) on ice. Next, protein levels were quantified using the Pierce™ BCA Protein Assay Kit (Sigma) and 30 μg of total protein from each sample was prepared with sample reducing buffer (2 M Urea, 2% SDS (sodium dodecyl sulfate), 0.125 M Tris HCl, 0.1M DTT (dithiothreitol) and bromophenol blue) at a ratio of 3:1 (lysate: reducing buffer) and resolved on an SDS gel with Tris/Glycine/SDS gel running buffer. The samples were subsequently transferred onto nitrocellulose membranes (BioTrace NT, Pall Life Sciences, New York, NY, USA) using a Transblot apparatus (Bio-Rad) and blocked using 1:1 Odyssey® blocking buffer (LI-COR): 1X PBS prior to probing with mouse anti-E-cadherin mAb (clone 36/e-cad, BD Biosciences), mouse anti-vimentin mAb (clone V9, Dako), and mouse Pan-actin mAb (clone ACTN05, Thermo Scientific). Membranes were then scanned on the Odyssey imaging system (Li-Cor, Lincoln, NE, USA) to obtain a visual representation of the amount of protein present in the samples.