ChIP/Liver harvest

Overview

This is a protocol for harvesting mouse liver tissue for chromatin immunoprecipitation.

This protocol is incomplete. More to come. ~ cmc 14:19, 6 December 2006 (EST)

Reagents

Buffer A (100mL total)

• 91mL of H2O
  
• 1.5mL of 1M Hepes
  
• 2mL of 3M KCl
  
• 300µL of 5M NaCl
  
• 40µL of 0.5M EDTA
  
• 100µL of 0.5M EGTA
  
• 16.2µL of 2.1M Sucrose
  
• 7.5µL of 2M BME
  
• 5mL of 2.5M Glycine (to make 2.5M stock- 187.5 g/L)
  
  • A stock of Liver Buffer A can be made without BME or Glycine and stored at room temp. Add BME and Glycine before use.

Buffer B (100mL total)

• 1.5mL of 1M Hepes
  
• 2mL of 3M KCl
  
• 300µm of 5M NaCl
  
• 20µm of 0.5M EDTA
  
• 20µm of 0.5M EGTA
  
• 100mL of 2.1M Sucrose
  

Other

• Avertin (Anesthesia)
• PBS
  
• PBS/1% Formaldehyde
  
• 2.5M Glycine

Supplies

Surgical Instruments

• Scissors
• Tweezers
• Bulldog clamp
• String

Other:

• 50mL conical tubes (1 per Liver)
• 2 of 10cc syringe
• 1 of 3cc syringe
• 26g needle
• Angiocatheter
• Ice with bucket

Procedure

Harvest

Total Time= ~20min/liver

• Open anaesthetized animal posterior to anterior
• Tie a loose knot of string around the superior vena cava in between the diaphragm and top of liver
• Tie a loose knot of string around the inferior vena cava
• Insert catheter into inferior vena cava from posterior to anterior and secure with bulldog clamp making sure that the end of the catheter is anterior to the kidney junction
• Screw 10cc syringe with PBS onto catheter
• Tighten both knots and cut the portal vein
• Perfuse with PBS very slowly. (3-5 minutes)
• Switch syringes and slowly perfuse with 10cc PBS/formaldehyde
  • The total time for both syringes of perfusion should be 5-10 minutes
• Cut diaphragm and remove liver
• Put liver into 50mL conical tube cap and dice into little pieces
• Incubate liver for 10 minutes and then add to 6mL glycine solution on ice

Notes:

• Keep Buffer A on ice and PBS and PBS/formaldehyde in 30o water bath
• Do not use mice older than 3 months or if they are sick
• Harvest even numbers of animals- 2, 4, 6

Hepatocyte Purification

• Pour minced liver into dousing apparatus and rinse beaker with Buffer A and add to dounce
• Dounce until liquefied
• Filter through plastic filter and wash with Buffer A
• Gently layer onto a 1.5mL cushion of 1:1 Buffer A and B in a Corex tube
• Centrifuge in fixed-angle rotor at 10,000rpm for 10min at 40
• Decant and discard supernatant
• Gently resuspend pellet in 5-10mL PBS
• Layer onto a 1.5mL cushion of 1:1 Buffer A and B in a Corex tube
• Centrifuge at 5,000rpm for 10min at 40
• Gently decant and discard supernatant
• Resuspend pellet in 3mL of sonication buffer or resuspend pellet in 5mL of PBS or TBS, transfer to 15mL conical tube, centrifuge, decant supernatant and freeze pellet in -800

Notes:

• Purification protocol should be done in the cold room on ice

Notes

• Please contribute user notes!

References

• (Young Lab)

Contact

• cmc