May 25, 2023
Intrapancreatic injection surgery
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Maria Jimenez Gonzalez, Rosemary Li, Sarah Stanley 2023. Intrapancreatic injection surgery. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnx47pl5d/v1
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: In development
We are still developing and optimizing this protocol
Created: January 09, 2019
Last Modified: May 25, 2023
Protocol Integer ID: 19127
Keywords: Intrapancreatic injection, Cholera Toxin Beta
Funders Acknowledgement:
SPARC
Grant ID: OT2OD024912
Abstract
This is a protocol for performing intrapancreatic injection of Cholera Toxin Beta (CTb) in mice.
Materials
MATERIALS
1 g LidocainebiorbytCatalog #orb341946
100 g Povidone IodinebiorbytCatalog #orb322666
BuprenorphinCatalog #961425
Isoflurane (Forane)BaxterCatalog #1001936040
Cholera Toxin Subunit B (Recombinant) Alexa Fluor™ 488 ConjugateThermofisherCatalog #C34775
Safety warnings
Wear Protective Personal Equipment (masks, labcoat, gloves) when working with animals. Isoflurane is an eye and skin irritant and central nervous system. Always include a propper scavenging system when utilizing it.
Before start
-This study was approved by the Institutional Animal Care and Use Committee of Icahn School of Medicine at Mount Sinai
-Clean and sterilize properly all surgical tools needed.
Preparing surgery area
Preparing surgery area
- Prepare the surgical space by placing the absorbant pad and the inhalatory anesthesia. Write down the mouse weight.
- Anesthetize the animal by inducing anesthesia with 3% Isofluorane. Once anesthesia is induced, place animal in supine position and keep anesthesia at 1-1.5%.
- Apply eye lubricant (eg. Lacri Lube). Subsequently apply hair removal cream (eg. Nair) in the area of incision (left abdominal area). Apply Povidone/Yodine in the incision area. Apply Lidocaine afterwards. Apply Ethanol swab. Now the surgical area is ready for incision
Surgery
Surgery
-Make a vertical incision of 1 to1.5cm, left side of the abdomen. Gently cut first skin, then abdominal wall. Cut a piece of gauze and wet it in sterile PBS, place close to the incision point. With the help of cotton tips or arruga forceps, expose the pancreas and place it in the gauze.
- 100 µg of CTb is dissolved in 5 µL Sterile PBS. With the use of a 5 µL Hamilton syringe, perform 5 injections of 1 µL volume each . In the case of AAV injection, total volume of injection should range between 5 µL -20 µL , depending on the concentration needed. Inject in several places of the pancreas (spread between head and tail of the pancreas). Every injection should be performed at a slow pace (5-10 seconds), waiting 10-15 seconds before removing syringe.
- Place the pancreas back to the abdominal cavity. Suture (2-3 sutures) the abdominal wall, using Vicryl Suture 4-0, 1/2c RB1 (or similar). Close the skin with an autoclip applier.
Post-surgery care
Post-surgery care
After surgery, inject Buprenorphin (SC, 0.05-0.1 mg/kg) and place animal back to its cage. Analgesia should be injected the 2 following days, twice a day. Monitor animal weight and behavior.
CTb expression
CTb expression
Harvest tissues of interest: Coeliac ganglia, Nodose ganglia, Dorsal Root Ganglia 1 week after injection of CTb. In the case of AAVs, harvest tissues 3-4 weeks after injection.