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Pipeline publication

[…] ckson ImmunoResearch Laboratories, Inc.) before mounting., All Z-stack acquisitions were done using a confocal microscope (SP5; Leica) with 20× and 40× lenses. The number of LCs per square millimeter of epidermis was determined by acquiring Z-stacks of immunolabeled epidermal sheets. A minimum of four Z-stacks (0.56 mm2/Z-stack) were acquired per epidermal sheet. The number of cells per Z-stack was enumerated and represented as cells per square millimeter. The distance between a langerin-GFP+ cell and its closest GFP+ neighbor (Ki67+ or Ki67−) was measured on a maximal intensity projection of Z-stacks of immunolabeled epidermal sheets. All image quantification and rendering was performed on MetaMorph (MDS Analytical Technologies) and Imaris (Bitplane) software., Langerin-GFP CCR2+/− or CCR2−/− mice of various ages (P2–P7) were anesthetized using 25 mg/kg ketamine, 5 mg/kg xylazine, and 0.8 mg/kg acepromazine injected intraperitoneally. Mice were kept at 37°C with oxygen (0.5 liter/min). Mice abdomens were placed on a custom-made tray-stage insert with a circular 2.5-cm hole covered with a coverslip. Images were acquired using an inverted confocal microscope (LSM 510; Carl Zeiss, Inc.) equipped with a 20×/0.75 Plan Apochromat objective. A thermostated chamber (Carl Zeiss, Inc.) was used to keep the microscope, mouse, tray, and objective at 37°C during the experiment. Z-stacks (with 1-µm Z-spacing) were acquired on the ventral skin (at least seven Z-stacks per mouse, 0.22 mm2/Z-stack), and the number of langerin-GFP+ cells per square millimeter was determined., Skin from langerin-GFP neonates (P4 and P7) and split ears of 10-wk-old mice were incubated in PBS 0.2% trypsin at 37°C for either 1 h or 25 min, respectively. The epidermis was peeled from the dermis and reincubated for 2 h in PBS with 1 mg/ml collagenase D (Roche). An epidermal cell suspension was obtained by mechanical dissociation of epidermal sheets on a 100-µm cell strainer (BD). GFP-expressing LCs were sorted (FACSAria II; BD). Sorted cells were washed with PBS and fixed in 70% ethanol at 4°c for 30 min. After […]

Pipeline specifications

Software tools MetaMorph, Imaris, Z-Spacing