TITLE: Cold Spring Harbor - Intraductal Injection 
AUTHORS: David Tuveson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">from Xiao et al., Nat Prot 2014, modified by KM. </div></div>

[STEPS]
?. [Anesthesia and Preparation]
Anesthetize mice with 2.5–3% (wt/vol) isoflurane by inhalation.
The inhalation should be closely adjusted, according to the breathing and heart rate of the mice during surgery. A typical infusion surgery takes 15-20 min. Unnecessary delays in the surgery should be avoided. (use Nair to remove hair from the lower chest and abdomen.)
?. [Anesthesia and Preparation]
Put the mouse onto the 37 °C warming pad. Immobilize the mouse on the head pad using surgical tape and Clean and disinfect the now hairless area with Betadine.
?. [Laparotomy]
Use scissors to incise the skin at the midline of the abdomen.
?. [Laparotomy]
Next, cut the abdominal muscle along the linea alba to create a midline upper abdominal laparotomy incision of 1.5 cm. Avoid injuring any underlying organs when performing the laparotomy.
?. [Laparotomy]
Clamp skin and peritoneum and rest the clamps on paper rolls.
?. [Exposure of the Pancreatic Duct]
Use wet cotton stick and ring forceps to touch the organs as follows:
?. [Exposure of the Pancreatic Duct]
Cover the duodenum with moistened gauze (with PBS) to keep the position and to avoid substantial heat loss and drying.
?. [Cannulation of the Duct]
A microclamp must be applied the common bile duct above the branch-point of the pancreatic duct, to prevent infusion into the liver.
When the first clamp is placed, clamping of the portal vein located immediately below the common bile duct should be avoided.
?. [Cannulation of the Duct]
Use a 30-gauge needle for B6J, 27-gauge for Nude and NSG to make a small hole opposite to the sphincter of Oddi, 1–2 mm away from the ampulla of Vater.
?. [Cannulation of the Duct]
Next, pass a 31(B6J) or 27(Nude/NSG)-gauge catheter or Hamilton syringe (50 µL, Model 705 SN SYR, Cemented NDL, 27gauge, 3 point style) through the small hole that has been created by the 30/27-gauge needle in the duodenal wall and then the sphincter of Oddi.
The tip of the catheter should be positioned at the origin of the pancreatic duct branch in the biliary-pancreatic duct. If the anatomy is unclear, the tip of the catheter can be placed just past the sphincter of Oddi.
?. [Cannulation of the Duct]
Clamp the papilla around the catheter with another microclamp at the sphincter of Oddi to prevent backflow or leaking during the infusion. The microclamp is of low pressure to avoid injury of the duodenum.
?. [Infusion]
Inject tumor volume with total amount  (50 ul of cell suspension)Rate /  manually
50 µl
50 µl
?. [Removal of the Cannula and Microclamps]
After the infusion is completed, remove the microclamp on the sphincter of Oddi and withdraw the syringe or catheter gently from the duct.
?. [Closure]
After infusion, close the hole in the duodenum created by the catheter with a 6-0 suture or a tissue glue for Nude and NSG.
For B6J, this suture does not seem to affect the experimental results, nor does it seem to affect survival of the mice.
?. [Closure]
Remove the microclamp on the biliary-pancreatic duct.
?. [Postoperative Care]
Return the mouse to its cage on a heated pad () until it fully recovers.
37 °C
?. [Postoperative Care]
Give ketoprofen at a dose of 5 mg/kg s.c. once per day continuously for 3 d after surgery for analgesia.
?. [Closure]
Return the duodenum to the right position and suture the abdominal muscles with a 4-0 suture using a running stitch.