Fluid Aspiration from Mouse Glomeruli: A Method to Aspirate Glomerular Fluid from Mouse Using Two-Photon Mediated Micropuncture
Transcrição
In the kidney, the glomerulus – a bundle of small blood vessels enclosed in the Bowman's capsule – initiates blood filtration and expels the waste fluid into the surrounding Bowman's space. Investigation of the glomerular fluid contents helps understand renal physiology.
To aspirate the glomerular fluid, prep an anesthetized mouse with its kidney surgically exteriorized. Now, inject fluorescent-dextran dye into the blood vessels behind the mouse's eye orbit. Fluorescent-dextran molecules move through the bloodstream and reach the kidney.
Due to the dextran’s high molecular weight, the fluorescent dye gets retained within the glomerulus, imparting it a green color. Under a two-photon microscope, which allows visualization of deeply seated structures, locate a suitable glomerulus to obtain adequate access to the glomerular fluid.
Next, align a red fluorescent nanoparticle-coated micropipette tip with the kidney's lateral axis. Using a micromanipulator, advance the pipette tip to penetrate the kidney. As the tip reaches the target glomerulus, set the desired aspiration rate to aspirate the glomerular fluid from the Bowman's space.
Absence of green fluorescence inside the red pipette tip indicates lack of bleeding and confirms successful glomerular fluid aspiration. Subsequently, retract the pipette and store the fluid in an appropriate buffer for further analysis.
