Computational protocol: Keratins regulate colonic epithelial cell differentiation through the Notch1 signalling pathway

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Protocol publication

[…] Protein samples were homogenized on ice in homogenization buffer (0.187 M Tris-HCl, 3% SDS, 5 mM EDTA, pH 6.8). Protein concentration was measured with BSA protein assay reagent kit (Thermo-Scientific, IL, USA), and samples were normalized and diluted in Laemmli sample buffer, heated at 95 °C for 3–5 min and 10 μg protein/sample was loaded in each well on 7–12% SDS-polyacrylamide gels. Prestained molecular weight markers (Bio-Rad) were loaded on each gel, and the molecular weights of the analysed bands are indicated on the right side on every blot/panel in the figures where kD refers to kilodalton. Rabbit anti-β-actin (Cell Signaling, MA, USA), rabbit anti-phosphohistone H3 (Cell Signaling), rabbit anti-histone H3 (Abcam, UK), rat anti-Hsc70 (Stressgen Bioreagents, MI, USA), rabbit anti-synaptophysin (Abcam), mouse anti-villin (Abcam), rabbit anti-full length Notch c-20 (Santa Cruz), rat anti-Troma I (Developmental Studies Hybridoma Bank, IA, USA), rabbit anti-K8 (273) and rabbit anti-K18 (275; John Eriksson, Finland) and mouse anti-cytokeratin 4.62 (K19, Sigma-Aldrich, CA, USA) were used as primary antibodies. Anti-rabbit IgG (Promega, WI, USA), anti-rat IgG (GE Healthcare and Cell Signaling) and anti-mouse IgG (GE Healthcare) were used as secondary antibodies. ECL (GE Healthcare) and ECL plus (Perkin Elmer, MA, USA) were used for detection of blots on X-ray films (Super RX, Fuji Corporation, Japan). Individual bands were analysed and normalized to the loading controls Hsc70 or actin using the ImageJ Software (NIH, MD, USA). [...] Colonic 7-μm sections of PC and DC were fixed in 1–4% paraformaldehyde (Sigma-Aldrich. CA, USA). Human Caco-2 cells were cultured and grown as described above and fixed at −20 °C for 10 min with methanol and for 1 min with acetone. Cells of tissues were stained as previously described. Primary antibodies used were rat anti-Troma I (Developmental Hybridoma Bank, NIH, MD, USA), rabbit anti-K8 (273) and rabbit anti-K18 (275), rabbit anti-full length Notch c-20 (Santa Cruz), mouse-anti Notch1 (A6; Thermo Scientific), rabbit anti-Hrt1 (Hey1, Santa Cruz), rabbit anti-phosphohistone H3 (Cell Signaling) and rabbit anti-synaptophysin (Abcam). Secondary antibodies used were donkey anti-rabbit conjugated with Alexa Fluor 488 (Invitrogen) and goat anti-rat conjugated with Alexa Fluor 546 (Invitrogen). Draq5 (Cell Signaling) or DAPI (Invitrogen) were used to stain DNA, and Phalloidin (Invitrogen) to stain F-Actin. ProLong gold antifade reagent (Invitrogen) was used for mounting coverslips. Leica TCS SP5 confocal microscope with dry objective HC Plan Apo × 20/0.70 NA ( and ) and a photomultiplier tube was used for image detection at room temperature using the Leica LAS AF acquisition software. Zeiss LSM780 confocal microscope with × 63/1.2 water objective was used for image detection (, , , ). Zeiss Zen image software was used for image analysis. Heatmap images () from confocal microscopy data were created with the BioImageXD software. All images in the individual panels were acquired with the same settings and adjusted for brightness and contrast identically using Photoshop CS5 (Adobe, CA, USA).Immunohistochemistry staining on paraffin-embedded colon sections was performed at the Department of Pathology, University of Turku Hospital. Sections were fixed in 4% PFA, incubated with rabbit anti-cleaved Notch1 (Abcam) primary antibody and secondary antibody conjugated to HRP. Sections were analysed with the Panoramic 250 Slide Scanner and Pannoramic Viewer was used for image acquisition using × 20 () and × 40 () settings. […]

Pipeline specifications

Software tools ImageJ, BioImageXD
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Colitis