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Edited from a protocol kindly provided by Dr. Guo-Ping Shi
Procedure:
Day 1:
Preparation of beads for immunoprecipitation
The night before harvest: under hood using sterile technique:
- Prepare the commercially available magnetic cell isolation beads: use 50 µl beads per mouse harvested.
- Wash beads in 1.7 ml eppendorf tube by suspending beads in 0.1% BSA (diluted in 1x PBS and sterile vacuum filtered under hood), by placing tube on small magnetic separator and aspirating off supernatant. Repeat this step another 2X.
- Suspend beads in 0.1% BSA (2 ml per 50 µl beads used) in a round bottom 15 ml polystyrene tube.
- Add anti-mouse PECAM-1; 5 µl per 50 µl beads and incubate on rocker at 4ºC overnight.
Day 2:
Preparation of the mouse and organs:
- Euthanize mouse using CO2 chamber.
- Submerge mouse in 70% ethanol.
- Working as sterilely as possible remove heart, lung, liver, spleen and brain using autoclaved instruments. Store organs on ice in DMEM high glucose supplemented with 5 ml penicillin/streptomycin.
- Prepare Type 1 collagenase:
Dilute 2 mg of collagenase per ml of 1% BSA.
Add 1 µl of 1M CaCl2 and 1 µl of 1 M MgCl2 per ml. - 25 ml of collagenase is required per mouse sample. Collagenase must be sterile vacuum filtered in hood before use.
- Coat P100 dishes with gelatin, let sit in incubator for at least 30 min, plates must be completely dried before cells are applied.
![]() | Following steps should be carried out under hood using sterile technique |
- Move organs to a petri dish and clean off fat or excess tissue.
- Mince organs using 2 autoclaved razor blades. Do not mince longer than 1 min.
- Transfer minced organs to a 50 ml tube containing 25 ml collagenase and let incubate in 37ºC water bath for one hour occasionally shaking mixture.
- Fix a cannula to a 60 ml syringe and titrate sample three times.
- Pipette mixture through a 70 µm disposable cell strainer into a fresh 50 ml tube.
- Centrifuge 50 ml tube at 1300 rpm for 5 min at 4ºC.
- Aspirate supernatant without disrupting pellet.
- Re-suspend pellet in 25 ml of 0.1% BSA and again centrifuge at 1300 rpm for 5 min at 4ºC.
- Aspirate supernatant and re-suspend pellet in 1 ml of 0.1 % BSA.
- Wash PECAM incubated beads by placing polystyrene tube on large separator and aspirating supernatant, refill with 10-15 ml of 0.1% BSA. Repeat this step two additional times.