Computational protocol: Increased Abundance of M Cells in the Gut Epithelium Dramatically Enhances Oral Prion Disease Susceptibility

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Protocol publication

[…] For morphometric analysis, images were analysed using ImageJ software (http://rsb.info.nih.gov/ij/) as described on coded sections []. Background intensity thresholds were first applied using an ImageJ macro which measures pixel intensity across all immunostained and non-stained areas of the images. The obtained pixel intensity threshold value was then applied in all subsequent analyses. Next, the number of pixels of each colour (black, red, green, yellow etc.) were automatically counted. For these analyses, data are presented as the proportion of positively-stained pixels for a given IHC marker per total number of pixels (all colours) in the specific area of interest (eg: SED, FAE, LP etc.). In each instance, typically 3–6 images were analysed per mouse, from tissues from multiple mice per group (n = 4–8 mice/group). Full details of all the sample sizes for each parameter analysed are provided in every figure legend. [...] Mice were given a single oral gavage of 2x1011 of Fluoresbrite Yellow Green labelled 200 nm microbeads (Polysciences, Eppelheim, Germany) in 200 μl PBS. Mice were culled 24 h later and Peyer’s patches and small intestine segments were snap-frozen at the temperature of liquid nitrogen. Serial frozen sections (6 μm in thickness) were cut on a cryostat and counterstained with DAPI. Images of SED from three Peyer’s Patches (duodenal, jejunal and ileal) and 8 LP areas per mouse (n = 3–4 mice/group) from 3 non-sequential sections (total 21–31 SED, or 24 LP areas per mouse studied) were typically acquired using a Nikon Eclipse E400 fluorescent microscope using Micro Manager (http://www.micro-manager.org). For example, each Peyer’s patch was trimmed until at least one SED region was visible and 20 sections collected. The 1st, 10th and 20th sections were then analysed. Tissue auto-fluorescence was subtracted from displayed images using ImageJ, the size of the area of interest in each section was then measured and the number of beads determined using the cell counter function in ImageJ and the bead density calculated. […]

Pipeline specifications

Software tools ImageJ, μManager
Application Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens