Préparation de la collagénase Peser la collagénase dans une mémoire tampon de 50 ml et ajouter l'isolement coniques pour rendre la concentration finale, comme indiqué dans le tableau ci-dessous. Dissoudre la collagénase par vortex. (12 + 2 souris souris extra = 14 souris x 5 ml / souris = 70 ml de solution de collagénase total) (70 ml x 0,5 mg / ml ou 0,3 mg / ml = 35 mg ou 21 mg de collagénase P total) <table…
Materials
Material Name
Type
Company
Catalogue Number
Comment
Dithizone
reagent
Dissolve 5mg Dithizone in 5 ml of DMSO in a 50 ml conical tube. Add 45 ml of normal saline and mix gently. Filter with a 60 cc syringe and a 0.22 “m syringe filter into a new 50 ml tube. Store at 4 OC, until use. Add 1 ml to each 35 mm counting Grid-plate, when needed.
PBS
Buffer
HBSS
Buffer
Isolation soln.
Buffer
HBSS containing 10 mM of HEPES, 1 mM of MgCl2, 5 mM of Glucose, pH 7.4
Wash soln.
Buffer
Isolation buffer containing 1mM CaCl2
Collagenase P
Boehringer Mannheim-Roche
1213873
Dissection tools
Hemostat clamp (n=2), Forceps (n=2), Scissors (for skin incision an for taking out pancreas from adjacent tissue)