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) Buffer B (100mL total) Other
 * 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.
 * 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
 * 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

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

Contact

 * cmc