Light-sheet fluorescence microscopy of ECi cleared organs. Optical clearing via ECi allows LSFM of soft and hard murine organs, as bones (calvaria, left), kidney (middle) and heart (right). While autofluorescent signals provide detailed information of general tissue composition, specific antibody-labeling enables the visualization of defined cell types, as e.g. endothelial staining (CD31). (By Anika Klingberg, Publication)
PMA-induced NETosis of human neutrophils. 2-Photon imaging of NETosis events induced via PMA-stimulation. The extracellular traps are stained with Sytox-Orange, while the activation of neutrophils is indicated via the expression of CD66b on the cell surface. (By Anika Klingberg)
Microglia in the brain of a CX3CR1-GFP mouse. In-situ 2-Photon imaging of a CX3CR1-GFP mouse brain shows GFP+ microglia in the tissue below the meninges (SHG) and the brain vasculature (CD31). (By Anika Klingberg, Publication)
Visualisation of NETs by bicoloured dSTORM. Human Neutrophils were stimulated with PMA for 3 hours at 37°C. Afterwards, the cells were fixed with 4% PFA. Histones H1 and Neutrophil Elastase were stained with antibodies. Consequently the MEA-buffer was added, which brought the fluochromes in a blinking state. (By Alexandra Brenzel and Jacqueline Heinen-Weiler)
Visualisation of NETs with central zoom in via SEM (= Scanning Electron Microscopy). Human Neutrophils were stimulated with PMA and E.coli added. They were incubated for 3 hours at 37°C to induce NETs, which caught the E.coli. (Inlens 50X, SE2 1.01KX, SE2 29.27KX) (By Jacqueline Heinen-Weiler)
Light-sheet fluorescence microscopy of ECi cleared kidneys. Optical sections of an ECi cleared and LSFM-visualized entire murine kidney show endothelial structures (CD31), as the interlobar vessels and glomerular capillary tufts. (By Anika Klingberg, Publication)
Visualisation of NETs via SEM (= Scanning Electron Microscopy). Human Neutrophils were stimulated with PMA and E.coli added. They were incubated for 3 hours at 37°C to induce NETs, which caught the E.coli. Afterwards the image was colored using Photoshop (Adobe). (By Jacqueline Heinen-Weiler)
In vitro hyphal cell wall staining of the fungus Aspergillus fumigatus.Aspergillus fumigatus tdTomato is stained with the fluorophore coupled antibody JF5 Dylight650 and the nucleus with DAPI. The fungus was grown for 16h after conidia seeding. (By Djamschid Solouk)
Combined confocal and 2-photon laser scanning microscopy of glomerular capillary tufts. Sequential confocal and 2-Photon laser scanning microscopy allows the visualization of endothelial structures (CD31) in healthy (left) and NTN-affected (right) glomerular capillary tufts in combination with SHG signal of the Bowman’s capsule in the autofluorescent kidney tissue (Scalebars = 20 µm). (By Anika Klingberg, Publication)
Alveolar macrophages in the lung. In-situ 2-Photon imaging of a MacBlue-eCFP mouse lung shows CFP+ alveolar macrophages in the alveoli of the lung (SHG). (By Sophie Henneberg)
The Catchup mouse is a novel animal model for highly neutrophil specific expression of the red fluorescent protein tdTomato. This specificity is achieved by modulation of the Ly6G (human homolog: CD177) locus, which is highly specific for neutrophil granulocytes in mice. Therefore, this mouse model not only allows intravital microscopy or flow cytometry of untouched and unstained neutrophils in various health and disease models, but, due to the features of the Cre-lox contruct, opens up the unique opportunity for a highly specific genetic modification of murine neutrophils. (For the original publication see: Hasenberg et al. Catchup: a mouse model for imaging-based tracking and modulation of neutrophil granulocytes)
How does the Catchup mouse work?
As Ly6G is a marker selectively expressed by murine neutrophils, it was considered a suitable target. The first step was the generation of a mouse line in which the first exon of Ly6g is replaced by a knock-in allele encoding the Cre recombinase (see also: Cre-lox system) and the fluorescent protein tdTomato separated by a self-splicing T2A peptide. (Figure 1) Heterozygous mice express both tdTomato and Ly6G, while homozygous mice lack Ly6G expression completely.
Figure 1. Catchup, a mouse model for the genetic manipulation of neutrophils. Targeting strategy for the generation of Catchup mice. Ex, exon.
For laboratories and institutes interested in our mouse line:
The Catchup mouse model is available for shipping from our institute in Essen, Germany. If you are interested in obtaining this mouse line for your research, do not hesitate to ask Prof. Dr. Matthias Gunzer (matthias.gunzer@uni-due.de) directly. He will refer you to the people responsible for mouse shipping, who will help you regarding all arising questions.
