(modified from Kirit Bhatt/Geoff Gurtner lab Stanford)
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Reagent	Manufacturer	Supplier
Avertin	lab stock	lab
Flunixin 50 mg/ml	Henry Schein Co., various others	Fisher NC9443026
Flunixin 0.5 mg/ml working
Ampicillin 10 mg/ml
antibiotic ointment
Isoflurane	Henry Scheine Co., various others	Fisher
electric clipper		lab
depilatory agent	Nair	CVS, etc.
standard surgical prep	various	lab
4/6/8/10 mm punch biopsy instruments	Henry Schein Company  Miltex Instrument Company, Inc. or Sklar Instrument	Fisher
silicone sheet 0.5 mm thick  JTR-S-0.5	Grace Bio-Laboratories, Bend, OR	Grace Bio-Laboratories, Bend, OR
Krazy Glue	Elmer's, Inc., Columbus, OH	Staples, Fisher
6-0 nylon sutures	Ethicon, Sommerville, NJ	Fisher
Mastisol	Ferndale Labs, Ferndale, MI	Fisher
Tegaderm	3M, St. Paul, MN	Fisher
telfa	Henry Schein Company	Fisher

Preparation of silicone rings:

1. Using a 10mm (or 8 mm) punch biopsy instrument, cut discs from silicone sheet.
2. Using 6mm (or 4 mm) punch biopsy instrument, remove centers from silicone discs to form rings.
3. Remove plastic backing (both sides), clean or immerse rings in 70% EtOH and remove to sterile surface to air dry. Do not contact dust, lint, etc. which will adhere avidly to ring surface.
   

Preparation of wounds and placement of rings:

1. Anesthetize mouse with Avertin.
2. [Optional: ampicillin/saline 15 mg/kg sc from 10 mg/ml stock (1.5 µl/g body weight).]
   
3. Remove hair from dorsum of mice (from base of ears to tail and well below each laternal midline) using clippers and Nair. Wipe off hair and Nair with damp cloth. Rinse with warm running water as needed and pat dry. Removing all hair over a wide area is essential for splint adhesion. Hair removal is conveniently done one day prior to wounding.
4. Wipe dorsum with 70% EtOH. Betadine sterile prep. Sterile drape.
   
5. Use a 4mm (or 6mm) punch biopsy instrument to score the skin (do not use it to cut through the skin - it will cut through the muscle as well). 2 wounds/WT mouse; 4 wounds can be placed on larger mice. Wounds placed more caudally can survive better because they are less easily accessed bythe mouse.
   
6. Using sping or Iris scissor, cut and remove the scored skin and underlying panniculus carnosus to create full-thickness wounds.
7. Grasp a ring with forceps, dip one surface in ample Krazy Glue on a clean surface, center ring over the wound and apply light pressure to ensure even contact.
8. [Optional and not clearly an advantage: Secure outer edge of rings to skin using 6-0 Nylon interupted sutures. This usually requires about 8 evenly spaced sutures.]
9. Apply antibiotic ointment to wound bed and cover with Telfa rectangles cut to cover wound and ring.
10. Apply Mastisol to skin surrounding rings. Cut Tegaderm to size and place over wounds and rings.
11. Flunixin first dose 2.5 mg/kg, sc (100 µl 0.5 mg/ml for 20 g mouse)
    
12. Allow recovery under warming lamp. Ideally, mice should be housed in separate cages. Otherwise they bite each other's rings, sutures, and wounds.

Monitor mice, results:

1. Standard postop care as per AEP.
   
2. Inspect rings regularly (i.e., QOD or QD prn) for signs of inflammation and infection. Replace sutures as needed since rings may come off (mice biting rings, rubbing rings on cage, etc.)
3. Under isoflurane anesthesia at appropriate intervals remove Tegaderm/telfa, photograph to document wound area, and apply fresh Tegaderm/telfa.

Harvest Tissues, Analyze:

1. Euthanize mice at appropriate times and process wound tissue and surrounding skin for histology, immunohistochemistry, immunofluorescence, electron microscopy, RNA or protein isolation, etc.
2. CO₂ narcosis->cardiac puncture blood to 1.5 ml µfuge tube->serum. Thoracotomy.
3. Dissect wound skin, fix on paper toweing (SQ against toweling) in cold 2% PFA/PBS x 2 hr->cold 8% sucrose/PBS->18% sucrose/PBS->shake ON->OCT blocks->H&E, IHC (CD31, other epitopes). Alternative: bissect 2 hr-fixed tissue->process one half for OCT blocks as above and place the other half in formalin or PBS->continue fixation->wax blocks (HDFCC Core facility)->H&E.
4. Quantify wound area from gross images and epithelial gap, granulation tissue and  vessel density from H&E and IHC.