Material
| Instruments | Solutions |
| 1 Curve Scissor | 50 ml tube 70% Etoh |
| 1 Straight Scissor | Etoh Spray bottle |
| 1 Bone Scraper1 | bottle of PBS |
| 2 Forceps (1 medium, 1 fine) | 5 10 cm plates |
Sacrifice Animals
Place mice in a bag and fill bag with CO2 - 3-5 minutes, alternatively Dry ice method
Pour Betadine over head
Spray animals with Etoh
Cut Skin sagitally from back of neck towards nose
Remove calvaria with scissors-place in PBS on ice in a petri dish
Scrape soft tissues off calvaria macroscopically and microscopically (total of 2 times)
Place in PBS
Mince with scissors into small pieces
Wash 2-3× in successive dilutions of Betadine:PBS and finally with PBS
Place in Digestive media in a conical tube
(75 ml DMEM -alpha , 0.150g Dispase II, 0.075 g Collagenase A)
Shake at 37℃ for 10 minutes - Discard first round of digestion - too many fibroblasts and unwanted cells - collect the subsequent supernatants
Wash and spin (1000 rpm) 3× with PBS to remove enzymes
Plate in Media with serum (10%)
