Collagenase Voorbereiding Weeg de collagenase in een 50 ml conische en voeg isolatie buffer aan de uiteindelijke concentratie, zoals aangegeven in de onderstaande tabel te maken. Los van de collagenase door vortexen. (12 muizen + 2 extra muizen = 14 muizen x 5 ml / muis = 70 ml in totaal collagenase oplossing) (70 ml x 0,5 mg / ml of 0,3 mg / ml = 35 mg of 21 mg collagenase P totaal) <table style="BORDER-RIGHT: #ddd 1px solid; BORDER-TOP:…
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)