Leica Microsystems
Leica TCS SPE Confocal Microscope
TCS SPE
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My project involves elucidating the roles of Eps15 Homology Domain containing protein 4 (EHD4) in the kidney. EHD4 is a member of the EHD family of proteins whose role is to regulate endocytic recycling. It is abundantly expressed in the kidney, specifically the inner medullary collecting duct (IMCD). IMCD is responsible for the proper maintenance of water balance of the body and we have recently observed that mice that lack EHD4 develop pronounced diuresis. Forward trafficking of the water channel Aquaporin2 is a key regulatory step in maintaining water homeostasis by the IMCD and since we observed a defect in water balance in these knock out mice, we hypothesized that in absence of EHD4, forward trafficking of AQP2 is defective.
Z-stack imaging of cells
mpkCCD c-11 cell line
Cells were grown on filters in serum/hormone free media with 0.1nM dDAVP applied on the basolateral side before transfecting them with either non-targetting or EHD4 siRNA. 48hrs after transfection, 1nM dDAVP was added on the basolateral side and cells were treated for immunofluroscence after 24 hrs.
NA
dDAVP is a stable agonist of the Anti-diuretic hormone that results in increased forward trafficking of AQP2 in mpkCCD cells. In absence of EHD4 this effect of dDAVP was absent. This observation was derived from the three dimensional scanning of the fixed cells. More AQP2 was retained in sub apical vesicles in the absence of EHD4.
3D viewing of the cells by performing Z-stacks
None
Z-stacking can be done
Expensive; not user friendly.
Good option for doing Z-stacking.
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