*2.2. Mouse HSC Characterization*

D4 or P1 mHSC cultures were analyzed for auto-fluorescence upon excitation at 405 nm and emission at 450 nm to detect vitamin A-positive oil droplets using a LSM 510 confocal microscope (Carl Zeiss Microscopy Inc., Thornwood, NY, USA) and the incidence of positive cells was established by comparison to the number of cells observed with the same instrument by bright field microscopy. mHSC were fixed with 4% paraformaldehyde for 15 min at room temperature and incubated with anti-αSMA (1:500; Invitrogen, Waltham MA) or anti-reelin (1:200; R&D Systems, Minneapolis, MN, USA) for 1 h and developed with, respectively, Alexa Fluor 488 goat anti-mouse IgG (Invitrogen) or Alexa Fluor 568 donkey anti-goat IgG (Invitrogen). The frequency of αSMA- or reelin positive cells was determined by comparison to the number of cells that were positive for DAPI nuclear staining Quantitative real time polymerase chain reaction (qRT-PCR) was used to evaluate gene expression in primary mHSC within 4 days of plating or after passage to P1 as described below.
