Jul 14, 2020
Version 2
Cold Spring Harbor - Intraductal Injection V.2
- David Tuveson1
- 1Cold Spring Harbor Laboratory
- NCI PDMC consortium
Protocol Citation: David Tuveson 2020. Cold Spring Harbor - Intraductal Injection . protocols.io https://dx.doi.org/10.17504/protocols.io.bijnkcme
Manuscript citation:
Xiao, X., Guo, P., Prasadan, K., Shiota, C., Peirish, L., Fischbach, S., Song, Z., Gaffar, I., Wiersch, J., El-Gohary, Y. and Husain, S.Z., 2014. Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of adeno-associated viral vectors and/or cell-tagging dyes. nature protocols, 9(12), p.2719.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 14, 2020
Last Modified: July 14, 2020
Protocol Integer ID: 39246
Abstract
from Xiao et al., Nat Prot 2014, modified by KM.
Materials
MATERIALS
S&T Vascular Clamp (7mm)AgnthosCatalog #00396-01
KetoprofenSigma AldrichCatalog #K1751
STEP MATERIALS
KetoprofenSigma AldrichCatalog #K1751
S&T Vascular Clamp (7mm)AgnthosCatalog #00396-01
S&T Vascular Clamp (7mm)AgnthosCatalog #00396-01
Protocol materials
S&T Vascular Clamp (7mm)AgnthosCatalog #00396-01
S&T Vascular Clamp (7mm)AgnthosCatalog #00396-01
S&T Vascular Clamp (7mm)AgnthosCatalog #00396-01
KetoprofenMerck MilliporeSigma (Sigma-Aldrich)Catalog #K1751
KetoprofenMerck MilliporeSigma (Sigma-Aldrich)Catalog #K1751
S&T Vascular Clamp (7mm)AgnthosCatalog #00396-01
S&T Vascular Clamp (7mm)AgnthosCatalog #00396-01
KetoprofenMerck MilliporeSigma (Sigma-Aldrich)Catalog #K1751
Anesthesia and Preparation
Anesthesia and Preparation
2m
2m
Anesthetize mice with 2.5–3% (wt/vol) isoflurane by inhalation.
Note
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.)
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
Laparotomy
1m
1m
Use scissors to incise the skin at the midline of the abdomen.
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.
Clamp skin and peritoneum and rest the clamps on paper rolls.
Exposure of the Pancreatic Duct
Exposure of the Pancreatic Duct
30s
30s
Use wet cotton stick and ring forceps to touch the organs as follows:
Use Cotton stick to gently pull out the stomach and position a wet gauze (with PBS) on the stomach and other organs.
Rotate and stretch the duodenum and position it on the gauze to expose the biliary-pancreatic duct and its junction with the duodenum (the sphincter of Oddi), which appears pale.
Cover the duodenum with moistened gauze (with PBS) to keep the position and to avoid substantial heat loss and drying.
Cannulation of the Duct
Cannulation of the Duct
3m
3m
A microclamp must be applied the common bile duct above the branch-point of the pancreatic duct, to prevent infusion into the liver.
S&T Vascular Clamp (7mm)AgnthosCatalog #00396-01
Note
When the first clamp is placed, clamping of the portal vein located immediately below the common bile duct should be avoided.
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.
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.
Note
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.
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.
S&T Vascular Clamp (7mm)AgnthosCatalog #00396-01
Infusion
Infusion
2m
2m
Inject tumor volume with total amount 50 µL (50 ul of cell suspension)
Rate 50 µL / 00:02:00 manually
Removal of the Cannula and Microclamps
Removal of the Cannula and Microclamps
30s
30s
After the infusion is completed, remove the microclamp on the sphincter of Oddi and withdraw the syringe or catheter gently from the duct.
Closure
Closure
2m
2m
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.
Note
For B6J, this suture does not seem to affect the experimental results, nor does it seem to affect survival of the mice.
Remove the microclamp on the biliary-pancreatic duct.
Check if the tumor injection is performed correctly by checking blue dye in the pancreas
Return the duodenum to the right position and suture the abdominal muscles with a 4-0 suture using a running stitch.
Postoperative Care
Postoperative Care
5m
5m
Return the mouse to its cage on a heated pad (37 °C ) until it fully recovers.
Give ketoprofen at a dose of 5 mg/kg s.c. once per day continuously for 3 d after surgery for analgesia.
KetoprofenMerck MilliporeSigma (Sigma-Aldrich)Catalog #K1751