Vector Labs
VECTASHIELD Antifade Mounting Medium with DAPI
H-1500
My project involves understanding the physiological role of Eps15 Homology-domain containing protein 4 (EHD4) in the kidney. EHD4 regulates endocytic recycling and my current hypothesis is that in the principle cells of the inner medullary collecting ducts of kidney EHD4 the subcellular localization of the water channel Aquaporin-2 (AQP2) is regulated. The purpose of this experiment was to visualize the changes in the subcellular localization of AQP2 in the presence and absence of EHD4 when treated with 1nM desmopressin (desmopressin is V2 receptor agonist that upregulates exocytosis of AQP2).
Immunofluorescence
mpkCCD c-11 cell line
Cells were grown on filters and transfected with either scrambled or EHD4 siRNA. Desmopressin was added on the basolateral side. Cells were then fixed using ice cold dry methanol followed by permeabilization with 0.2% triton-X. Overnight incubation with goat anti-aqp2 was done after blocking. Secondary antibody was added the next day, followed by adding DAPI.
Must use it in dark. 10 ul or just a drop of the reagent will be sufficient for visualizing cells.
Good staining of the nuclei with this DAPI and the mounting media is also perfect.
None
Staining is good and the coverslip sets perfectly.
The dropper that comes with the kit does not work.
Good mounting media with DAPI