The overall goal of this procedure is to determine the obstructions of urinary tract junctions via methylene dye injection into kidneys. This method can help answer key questions in the kidney and lower urinary tract development field, such as uretal bladder connection. The main advantage of this technique is that it provides a simple method to visualize the urinary tract structure in worst case mass development enabling urinal tract development analysis.

To begin the experiment measure 0.1 gram of methylene blue powder. Dissolve the methylene blue in 10 milliliters of 1x phosphate buffered saline or PBS and vortex the mixture. Next filter the 10 milligram per milliliter methylene blue solution with a syringe filter to eliminate clogging during injection.

Then assemble a sterile scalp vein set with a three milliliter disposable syringe and fill the syringe with three milliliters of filtered methylene blue solution. Clean dissecting scissors and forceps with 70%ethanol. To collect perinatal embryos at E18.5 or E19.5 first euthanize pregnant mice using carbon dioxide inhalation.

Then perform cervical dislocation according to NIH guidelines. Next spray 70%ethanol on the ventral abdominal surface of the animal. Then using the sterile dissecting scissors and forceps open the abdominal cavity ventrally.

Lift the entire uterus and separate it from the body at the tips of the uterine horns. Then rinse the entire uterus with 1x PBS in a petri dish. Cut the uterus segmentally with dissecting scissors and remove the placental decidua with dissecting forceps to expose the embryos and yolk sacs.

Remove the yolk sac first and then remove the amniotic membrane with dissecting forceps to liberate the perinatal embryos. Decapitate the embryo with dissecting scissors. Pin the embryo down with its ventral surface up on a dissecting microscope equipped with a digital camera for imaging.

Collect its tail for genotyping. Using forceps carefully tear the skin to open the abdominal cavity of the embryo. Then carefully remove excess organs and tissues such as liver, stomach and intestines with forceps by cutting them or pulling them out to expose the kidneys, the ureters and the bladder that are located dorsally.

Absorb excess blood from the dissected embryo with sterile gauze pads then continue to dye injection. Remove bubbles in the needle and tubing by expelling methylene blue solution from the needle tip by hydrostatic pressure. Lift the syring containing the dye solution above the level of the needle tip to start flow.

And then lower the syringe to stop flow. Next insert the needle into the renal pelvis near the proximal ureter taking care not to disturb it once placed. Perform dye injection into a kidney to determine its urinary tract obstruction.

After insertion of one needle into the renal pelvis the success of this test a needle should be inserted into the renal pelvis towards the ureter neither passing through the kidney nor towards the blood vessel. Lift the syringe up about 20 centimeters to provide hydrostatic pressure and let 15 to 16 microliters of the dye solution flow. Then monitor the blue color of the dye starting first in the renal pelvis then in the length of the ureter and finally in the bladder lumen.

Place the syringe down to stop the dye flow and remove the needle from the kidney. Make a record of hydronephrosis of the kidney, hydroureter and the final position of the dye solution in a lab notebook. Finally perform injection of the contralateral kidney and allow the dye to flow for 20 seconds total.

Take images of the kidney, the ureter and the bladder traced with dye solution using the camera and imaging program connected to a stereo microscope. After injection into one kidney the methylene blue dye flows from the renal pelvis through the ureter and into the bladder resulting in a visible blue color in the ureter and bladder lumen. A stronger blue appears in the bladder lumen due to a longer flow about 20 seconds after injection of both kidneys with dye indicating the absence of urinary tract junction obstruction in the second urinary system.

When injecting an abnormally dilated hydroureter the final position of the dye solution is unclear. And ureterovesical junction obstruction or UVJO cannot be verified despite the absence of the dye in the bladder lumen because the abnormal hydroureter was damaged in the middle. While attempting this procedure it's important to remember to calculate the dye sac perinatal embryos to get intact urinary system.

And remove bubbles not to block the dye flow.