Quantifying Intestinal Inflammatory Monocytes

The whole small intestine was explanted, and 1 cm of the aboral end was cut and frozen in kryoTec freezing medium (Kryotec-Kryosafe GmbH, Hamburg, Germany). Five-µm slices were cut and fixed with acetone. Staining of inflammatory monocytes was performed with F4/80 antibody (AbD Serotec, Kidlington, UK) and secondary antibody goat anti-rat A647 (abcam plc. Cambridge, UK). DAPI (Thermo Fisher Scientific Inc) was used to counterstain cell nuclei. For histological analysis, images were recorded as 24-bit TIFF files on a Keyence BZ-9000® fluorescence microscope (Keyence Corporation, Osaka, Japan) with 20 × magnification, then saved using the BZ-II Image Viewer (Ver. 1.41, Keyence Corporation) and Analyzer (Ver. 1.42, Keyence Corporation). Cell segmentation was determined using a "positive cell detection" algorithm in QuPath (0.2.0-m4, University of Edinburgh, Scotland) with cell categorization by cytoplasm^{58 (link)}. Data were compiled using jupyterlab (1.2.5) with the following packages: Python (Ver. 3.6.8), Numpy (Ver. 1.18.1) and Pandas (Ver. 0.24.0). Each biological replicate was calculated by the mean of three technical replicates.

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Menz J., Hundt L., Schulze T., Schmoeckel K., Menges P, & Domanska G. (2021). Increased mortality and altered local immune response in secondary peritonitis after previous visceral operations in mice. Scientific Reports, 11, 16175.

Publication 2021

F4/80 antibody DAPI Keyence BZ-9000® fluorescence microscope

Acetone Antibody Biological Cell Cell nuclei Dapi Fluorescence microscope Goat Inflammatory Monocytes Python Replicate Small intestine

Corresponding Organization :

Other organizations : Universitätsmedizin Greifswald

Top 5 similar protocols

Variable analysis

independent variables

Explantation of the whole small intestine

dependent variables

Inflammatory monocyte staining with F4/80 antibody
Cell nuclei staining with DAPI

control variables

Kryotec freezing medium for sample preparation
Acetone fixation of 5-μm slices
Goat anti-rat A647 secondary antibody
Keyence BZ-9000® fluorescence microscope with 20× magnification
Cell segmentation and categorization using QuPath software
Data compilation using Jupyterlab with Python, Numpy, and Pandas

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