Computational protocol: Tubular Bridges for Bronchial Epithelial Cell Migration and Communication

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Protocol publication

[…] Time-lapse light microscopy of cells grown on 6-well plates was performed with an inverted microscope (Nikon Diaphot) and digital camera (Nikon D50) using 4x and 10x lenses over specified time periods. For immunofluorescence, cells were fixed for 20 min. with 4% paraformaldehyde. Cells were exposed to 0.2 M glycine for 10 min and incubated with 0.2% triton X-100 in phosphate buffered saline for 10 min. Goat serum (4%) in phosphate buffered saline with 1% bovine serum albumin was applied for 1 h at room temperature (RT). Primary antibodies were applied to the cells overnight at 4°C: Collagen IV (1∶50, AbCam); E-cadherin, Golgi marker, vimentin, CC10, MUC5AC, cytokeratin-5, smooth muscle α-actin (1∶50, Santa Cruz); CD31 (1∶50, Serotec); anti-acetylated α-tubulin (1∶50, Sigma). Secondary antibodies – alexa fluoro 488 and 594 (1∶50, Invitrogen) – were applied to the cells for 1 h at RT with or without rhodamine-phalloidin (1∶250, Invitrogen) for visualization of F-actin. Cells were mounted with VectaShield containing DAPI (Vector Labs). Image-iT™ LIVE Lysosomal and Nuclear Labeling Kit was used following the manufacturer's instructions (Invitrogen) for staining of lysosomes and nuclei. All images were obtained using a Zeiss LSM510 Laser scanning confocal microscope with a 25x lens or a Leica DMRA2 fluorescent microscope with 10x, 20x, 40x, and 100x lenses, and a Hamamatsu C4742-95 digital camera and MetaMorph software. ImageJ 1.42a software was used to generate 3D images from confocal image stacks of 50 to 100 consecutive 0.5 µm z-slices.Masson Trichrome straining (IMEB) was performed using Bouin's solution as a mordant overnight at RT. Cells were stained with Hematoxylin for 10 min, Biebrich's Scarlet Acid Fuchsin for 15 min, followed by Phosphotungstic/Phosphomolybdic Acid staining for 15 min. Aniline Blue stain was applied for 15 min. then cells were exposed to 1% acetic acid solution for 1 min, dehydrated through an ethanol series, cleared with xylenes for 5 min, and mounted with Cytoseal XYL (EMS). Photomicrographs were taken with a DC-70 digital camera (Olympus) on a Nikon Labphoto-2 microscope with 4x, 10x, 20x, and 40x lenses. [...] Data are expressed as standard error of the mean. Statistica 6.0 software was used to perform statistical analysis. Student's t test analysis was performed for data between 2 groups. ANOVA followed by Fisher's least significant difference post hoc test to assess intergroup significance was used to evaluate data among >2 groups. Significance was considered as a value of p<0.05. […]

Pipeline specifications

Software tools MetaMorph, ImageJ, Statistica
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Chemicals Oxygen