Preparação colagenase Pesar a colagenase em um buffer de 50 ml de isolamento ml e juntar para fazer a concentração final, conforme indicado na tabela abaixo. Dissolver a colagenase em vortex. (12 + 2 ratos ratos extra = 14 ratos x 5 ml / mouse = 70 ml de solução de colagenase total) (70 ml x 0,5 mg / ml ou 0,3 mg / ml = 35 mg ou 21 mg de colagenase total P) <table style="BORDER-RIGHT: #ddd 1px solid; BORDER-TOP: #dd…
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)