Fluid Aspiration from Mouse Glomeruli: A Method to Aspirate Glomerular Fluid from Mouse Using Two-Photon Mediated Micropuncture
Transcription
After confirming a lack of response to toe pinch, apply ointment to the eyes and use tape to immobilize the extremities of a 20- to 25-gram adult mouse. Use a depilatory cream to remove all of the hair on the left side of the animal, and use the spleen to locate the left kidney through the skin on the dorsal and caudal side of the spleen.
Make a 0.5-centimeter incision in the skin, followed by a smaller incision in the peritoneum, just large enough for the kidney to be pushed through. Extrude the kidney with gentle pressure and place a polysiloxane kidney stabilizer form around the tissue. Line the kidney up with the spacer such that the lateral-most surface of the kidney extends beyond the stabilizer by about 1 millimeter and fix the kidney to the form with cyanoacrylate adhesive. Glue a head plate to the stabilizer form and mount the head plate to mounting bars on the base plate.
Next, fill the well in the polysiloxane support with 1% agarose solution and hold a 10-millimeter coverslip on top of the form until the agarose is firm. Seal the coverslip to the head plate with glue and create a ring around the coverslip with dental cement. Then, inject 100 to 150 microliters of FITC-dextran retro-orbitally and quickly move the mouse and fixation plate to the two-photon microscope stage.
After manually focusing on the kidney surface, switch to the non-scanning two-photon mode and explore the imaging window to locate a target glomerulus that is greater than 30 micrometers below the coverslip and less than 400 micrometers from the lateral kidney capsule. Record the lateral and vertical distance to the target glomerulus. Then, record the x, y, and z stage coordinates for the glomerulus, and lastly, raise the objective focal point about 1 millimeter into the water column without changing the x and y stage coordinates.
Drive the pipette tip into the water column and turn on the DAPI excitation. Move the pipette in the X and Y dimensions to the point of maximal fluorescence of the tip. This will be the center of the objective. Change the excitation setting to red fluorescent protein and visualize the pipette with the ocular to allow precise centering in the ocular view.
Switch back to two-photon to find the pipette under the live two-photon view and place the tip precisely in the center of the image. This is the registration position. Then, save an image of the pipette and register the stage and the micropipette controller coordinates. Remove the pipette from the water column in the x-axis without moving the y and z axes, and move the pipette Z to the target glomerulus Z coordinate and the edge of the kidney.
Note the stage X and calculate the kidney edge pipette X using the offset from the registration stage, X. Increase the stage X to move the stage toward the pipette until the edge of the kidney is far to the left of the screen while remaining visible. Quickly advance the pipette to about 100 micrometers away from the kidney edge pipette X as just calculated. Increase the red gain and begin advancing the pipette tip slowly to the kidney edge under live two-photon imaging while monitoring the red pixel histogram.
Drive the pipette in the x-axis slowly to the glomerulus target pipette X, keeping an eye on the stage X. Upon reaching the glomerulus, document the position with a Z stack. With the pipette in position, set the micropump to inject 100 nanoliters of perfluorodecalin over 2 minutes to ensure patency of the pipette and to reduce confounding from pipette plugging during entry. After 4 to 6 minutes of filtration, set the micropump to aspirate up to 300 nanoliters at a rate of up to 50 nanoliters per minute.
