The overall goal of this procedure is to introduce cells into defined areas of the bladder wall. This is accomplished by first making an abdominal incision to expose the bladder. The next step of the procedure is to insert the bevel of the needle intramurally.
The final step of the procedure is to push the plunger of the syringe to inject the cells of interest into the bladder wall. Successful implantation of cells is marked by a well localized bleb that does not leak fluid and stays stable in size. The main advantage of this technique over existing methods like transurethral inoculation of mice with cells, is that it enables one to direct where the cells will implant themselves within the bladder.
Visual demonstration of li muscle is critical as the injection stay are hard to learn. Knowing the correct angle and the entry position of the needle into the bladder is essential to the technique success. Select the age, sex, and strain of mice based on needs of the experiment here.
Eight to 12 week old mice are used to begin clean the surface of the surgical table with soap and water. Next, wipe the surgical table surface with sideswipes or antiseptic wipes. Surgical instruments should be sterilized in an autoclave prior to surgery.
Wipe the instruments with 70%ethanol just prior to use and re-sterilized with a hot bead sterilizer between individual surgeries. Clean a 100 microliter Hamilton syringe attached to a 30 gauge needle by repeated aspiration of absolute alcohol, followed by sterilized phosphate buffered saline. Place an anesthetized animal in the supine position on a warm pad to help maintain normal body temperature.
Maintain a sufficient level of anesthesia by placing the animal snout in a nozzle containing vaporized isof fluorine. Next, remove the hair from the abdominal skin with clippers. Use a disposable sterile surgical drape to cover the anus to prevent fecal contamination during surgery.
Place a second surgical drape over the lower abdomen and expose a surgical window. Sterilize the surgical site with repeated applications of Betadine and alcohol using a dissecting microscope for magnification. Make a lower midline abdominal incision with scissors.
Use gentle pressure on the abdomen to expose the bladder. Since the bladder is usually partially or completely full pressure on the abdomen will preferentially push away other organs and allow the bladder to emerge the incision. If the bladder is full, apply gentle downward pressure at the dome to partially decompress it.
Fill the sterile Hamilton syringe with 50 microliters of the sample solution. Insert the tip of the needle with the bevel facing up into the wall of the bladder and inject the sample solution. A successful injection is marked by a well localized bleb that does not leak fluid and maintains a stable size.
After the injection close the incision with wound clips. Allow the master to recover on a warming pad One master. This technique can be done in 10 to 15 minutes if it is performed properly.
Success can be confirmed through histological analysis of the bladder at serial time points post injection.
