Total hepatic RNA was extracted with TriZol reagent (Invitrogen) according to a standard protocol. Mouse livers were fixed in 10% neutral buffered formalin or Methacarnoy (60% methanol, 30% chloroform and 10% acetic acid: v/v/v) overnight, and analyzed using standard histological techniques. IHC was performed using antibodies specific for BrdU (anti-mouse Dako, Carpinteria, CA), CD34 (anti-rat CD34, Cedar Lanes), vWF (Dako, A0082), anti-active caspase 3 (Cell Signaling), anti-PDGFRalpha (R&D AF1062) and, anti-PDGFRalpha (Cell Signaling). Detection of the primary antibody was carried out using the appropriate biotinylated secondary antibody (Vectastain, Burlingame, CA) and the peroxidase DAB kit (Ventana, Tucson, AZ). Nuclear incorporation of BrdU into liver cells was used to measure cell proliferation using the mouse on mouse (M.O.M.) kit (Vectastain) to detect NPCs and hepatocytes, and the number of mitotic figures were evaluated.
Label
biotin
Label protocol
For microarray hybridization, RNA was processed for hybridization to Affymetrix Mouse Gene 1.0 ST Array through a core facility in the Functional Genomics Laboratory in the Center for Ecogenetics and Environmental Health at the University of Washington according to the manufacturer's instructions.
Hybridization protocol
The standard hybridization protocol was used as recommended by Affymterix.
Scan protocol
GeneChips were scanned using the Affymetrix GeneArray Scanner 3000 through the University of Washington Functional Genomics Laboratory in the Center for Ecogenetics and Environmental Health (http://depts.washington.edu/ceeh/ServiceCores/FC1/FC1.html).
