TITLE: Mouse Pancreas Dissection and Fixation for Cryosectioning
AUTHORS: Islet and Pancreas Analysis Core 

[DESCRIPTION]
This SOP defines the method used by the Vanderbilt Diabetes Center Islet and Pancreas Analysis (IPA) Core for fixation and embedding of mouse pancreas.

[STEPS]
SECTION: Reagent preparation
1. Ketamine (15 mg/mL)/xylazine (3 mg/mL) working solution – Use 5 mL syringes with 18G needles. Label all bottles with additive, mix date, and expiration date of earliest-expiring ingredient.
SECTION: Pancreas Dissection and Fixation
1. 
SECTION: Pancreas Dissection and Fixation
1.1. 
SECTION: Reagent preparation
1.1. Pull 2 mL of xylazine and inject into a 10 mL bottle of ketamine.
SECTION: Reagent preparation
1.2. Pull 2 mL of ketamine/xylazine and inject into a 10 mL bottle of saline.
SECTION: Pancreas dissection and fixation
5. Inject mice intraperitoneally with ketamine/xylazine solution (1 μl/mg body weight) using a 1 mL syringe with a 27G needle. Place mice into a plastic bin with air holes. 
SECTION: Pancreas dissection and fixation
5.1. After 5-10 minutes, check to see if mice are fully anesthetized bygently pinching feet and tail. If there is a jerking or squeaking response, inject about 100 μL more of ketamine/xylazine solution, and wait a few more minutes. Continue checking for full anesthetization before beginning surgery.
SECTION: Embedding and cryosectioning
12. Coat a labeled cryomold with a thin layer of OCT compound and set aside.
SECTION: Reagent preparation
2. 0.1 M PBS (1L) – Dissolve 12.07 g Na2HPO4 (dibasic), 8.0 g NaCl, and 2.0 g KCl in 1L Milli-Q water.  This solution should be made fresh or can be sterile filtered, kept at 4 °C, and used within 1 month.

SECTION: Reagent preparation
3. 4% Paraformaldehyde (PFA) – Dilute one ampule (10 mL) 16% PFA in 30 mL 0.1M PBS. This solution should be made fresh the day of fixation. Approximately 13mL per pancreas is required.
SECTION: Reagent preparation
4. 30% Sucrose – Dissolve 15 g sucrose in 35mL 10 mM PBS. Solution can be placed on shaker to ensure sucrose fully dissolves.  Approximately 13 mL per pancreas is required.
SECTION: Reagent preparation
3.1. Pipet 13 mL aliquots into labeled 15 mL conical tubes (one per pancreas). Using analytical balance, record weight of each tube to nearest 0.01 g.
SECTION: Pancreas dissection and fixation
6. Place mouse face up on the surgical stage, tape limbs down, and wipe with 70% ethanol. Open the body cavity using forceps and scissors and locate pancreas by moving stomach and intestines to the side. Use two pairs of forceps to separate the pancreas from the duodenum and stomach, starting from the head. Remove the pancreas with the spleen still attached.

  
  

SECTION: Pancreas dissection and fixation
7. Place the pancreas in a 100 mm tissue culture dish containing 10 mL ice-cold 10 mM PBS. Dissect away the spleen and remove all fat tissue attached to pancreas.
 

SECTION: Pancreas dissection and fixation
8. Place pancreas into preweighed 15 mL tube with 4% PFA (step 3.1). Using analytical balance, record weight of tube containing pancreas to nearest 0.01 g.
SECTION: Pancreas dissection and fixation
9. Fix pancreas for 120 min on ice with gentle agitation using a shaker at low speed.

SECTION: Pancreas dissection and fixation
10. Decant fixative and wash pancreas 4 times in approximately 13 mL 0.1 M PBS for 30 min. Tubes should be kept on ice for washes with gentle agitation using a shaker at low speed.

SECTION: Pancreas dissection and fixation
11. Decant PBS and add approximately 13 mL 30% sucrose. Store 30 min at 4 °C to cryoprotect pancreas.

SECTION: Embedding and cryosectioning
13. Retrieve 15 mL conical tube with pancreas in 30% sucrose (step 11) and pour contents into a 100 mm tissue culture dish.
SECTION: Embedding and cryosectioning
13.1. Using fine forceps, pick up pancreas and blot excess sucrose from tissue using Kimwipes.
SECTION: Embedding and cryosectioning
14. Place the pancreas into the cryomold as shown below, using forceps to gently push the tissue to the bottom.

  
SECTION: Embedding and cryosectioning
15. Place cryomold with pancreas onto a layer of dry ice. Fill remaining volume of cryomold with OCT, covering the pancreas, and let freeze thoroughly on dry ice until OCT is opaque.
SECTION: Embedding and cryosectioning
17. Cut cryosections at 5-10 μm thickness and mount to Plus Gold charged slides for staining. For mass quantification, collect cryosections throughout the entire pancreas and to obtain at least 5 sections 200 μm apart.

 

SECTION: Embedding and cryosectioning
16. Wrap frozen cryomold in pre-labeled square of aluminum foil, place into a sealed plastic bag, and store at -80 °C until ready to cryosection.