Supplementary Experimental Procedures
Materials
Chemicals and reagents were from Sigma Chemical Co., St. Louis, MO and Invitrogen Corp.,
Carlsbad, CA; collagenase type 1 from Worthington Biochemical Corporation, Lakewood, NJ;
cell culture medium and supplements from Gibco BRL Life Technologies, Grand Island, NY;
hormones from Reprotech Inc., Rocky Hill, NJ or Upstate Biotechnology, Lake Placid, NY; and
FBS from Gemini Bio-Products, Woodland, CA.
Fixation conditions used for immunohistochemistry
Cell suspensions or cultured cells were fixed on cytospins or 2-well chamber slides by incubation
in ice-cold 4% paraformaldehyde for 10 min or cold acetone (-20ºC) for 5 min. After fixation in
paraformaldehyde, cytospins/chamber slides used for detection of cytoplasmic markers were
incubated in 0.1% Triton X-100 for 10 min.
Immunohistochemical detection of Thy-1, AFP, CK-19, albumin (Alb), E-cadherin, and
CXCR4
Thy-1, AFP, Alb, and CXCR4 detection. After fixation, cytospins were blocked in 2% donkey
serum, 1% BSA and 0.05% Tween. Cytospins were stained with mouse anti-Thy-1 (1:10; BD),
rabbit anti-AFP (1:100; LabVision), rabbit anti-Alb (1:100; ICN) or rabbit anti-CXCR4 (1:100;
Torrey Pines) as primary antibody. Secondary antibody was CyTM3-conjugated donkey antimouse IgG or CyTM3-conjugated donkey anti-rabbit IgG (1:100; Jackson).
CK-19 and E-cadherin detection. Cytospins/chamber slides were blocked in 2% goat serum, 1%
BSA and 0.05% Tween. Cytospins/chamber slides were stained with mouse anti-CK-19 (1:10;
Progene) or mouse anti-E-cadherin (1:50; BD) as primary antibody. Secondary antibody was
Biotin-conjugated goat anti-mouse IgG2 (1:50; Nordic), followed by incubation with
Streptavidin-CyTM2 (1:100; Rockland).
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Simultaneous detection of AFP/Thy-1, Ki-67/Thy-1, Alb/Thy-1, CD45/Thy-1, Alb/CD45, Ki67/AFP, CXCR4/AFP, and CXCR4/Thy-1/AFP
AFP/Thy-1, Ki-67/Thy-1 and Alb/Thy-1 double staining. Cytospins/chamber slides were blocked
in 2% donkey serum, 1% BSA and 0.05% Tween. After incubation with rabbit anti-AFP (1:100;
LabVision), rabbit anti-Ki-67 (1:800; LabVision) or rabbit anti-Alb (1:100; ICN) and mouse
anti-Thy-1 (1:10; BD) as primary antibodies, cytospins/chamber slides were stained with CyTM2conjugated donkey anti-rabbit IgG and CyTM3-conjugated donkey anti-mouse IgG (1:100;
Jackson) as secondary antibodies.
CD45/Thy-1 simultaneous detection. Cytospins/chamber slides were blocked in 2% goat serum,
2% rabbit serum, 1% BSA and 0.05% Tween. Cytospins/chamber slides were stained with
mouse anti-CD45 (1:50; Serotec) and mouse anti-Thy-1 (1:10; BD) as primary antibodies.
Secondary antibodies included Biotin-conjugated goat anti-mouse IgG2 (1:50; Nordic) and
TRITC-conjugated rabbit anti-mouse IgG1 (1:100; Rockland). For CD45 detection, cytospins
were stained with Streptavidin-CyTM2 (1:100; Rockland.).
Alb/CD45 double staining. After blocking in 2% goat serum, 2% donkey serum, 1% BSA and
0.05% Tween, cytospins were stained with rabbit anti-Alb (1:100; ICN) and mouse anti-CD45
(1:50; Serotec), CyTM3-conjugated donkey anti-rabbit IgG (1:100; Jackson) and Biotinconjugated goat anti-mouse IgG2 (1:50; Nordic) thereafter, followed by incubation with
Streptavidin-CyTM2 (1:100; Rockland).
Ki-67/AFP and CXCR4/AFP double staining. Cytospins/chamber slides were blocked in 2%
donkey serum, 1% BSA and 0.05% Tween. Cytospins/chamber slides were stained with rabbit
anti-CXCR4 (1:100; Torrey Pines) or rabbit anti-Ki-67 (1:800; LabVision) and sheep anti-AFP
(1:200; Nordic) as primary antibodies. Secondary antibodies included CyTM3-conjugated donkey
anti-rabbit IgG and CyTM2-conjugated donkey anti-sheep IgG (1:100; Jackson).
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CXCR4/Thy-1/AFP simultaneous detection. After blocking in 2% donkey serum, 1% BSA and
0.05% Tween, cytospins were stained with rabbit anti-CXCR4 (1:100; Torrey Pines), mouse
anti-Thy-1 (1:10; BD) and sheep anti-AFP (1:200; Nordic). Secondary antibodies included
CyTm2-conjugated donkey anti-rabbit IgG, CyTm3-conjugated donkey anti-mouse IgG, and
CyTm5-conjugated donkey anti-sheep IgG (1:100; Jackson).
Stromal cell-derived factor 1α (SDF-1α) detection in liver tissue
Five m sections of formalin-fixed/paraffin-embedded liver tissue were deparaffinized in xylene
and rehydrated by decreasing ethanol concentrations. Sections were treated by microwave
antigen retrieval for 15 min in 10 mM citrate buffer, pH 6. After blocking in 2% donkey serum,
1% BSA and 0.05% Tween, sections were stained with mouse anti-SDF-1α (1:50; R&D) as
primary antibody. Secondary antibody was CyTm3-conjugated donkey anti-mouse IgG (1:100;
Jackson).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blot analysis
RT-PCR analysis. Total RNA was extracted from fetal liver suspensions using Trizol reagent,
following the manufacturer’s protocol. RNA was reverse transcribed using SuperScriptTM III
reverse transcriptase and oligo-dt primers to synthesize cDNA (Invitrogen Corp.). Primers used
for these analyses are shown in Supplementary Table 1. cDNA was amplified by Ampli-Taq
Gold DNA polymerase (Applied Biosystems, Foster City, CA) for 10 min at 95˚C, followed by
20-40 cycles at 94˚C for 30 sec, 55˚C for 60 sec, 72˚C for 60 sec and a final cycle of 72˚C for 7
min (see Supplementary Table 1).
Western blot analysis for detection of AFP and CK-19. The proteins were resolved by 10%
reduced SDS-PAGE and transferred to nitrocellulose membranes using a Semi-Dry Transfer Cell
system (Bio-Rad Laboratories Inc., Hercules, CA). After blocking in 5% non-fat dry milk, the
membranes were incubated with rabbit anti-AFP (1:1000; LabVision) or mouse anti-CK-19
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(1:500; Progene). Secondary antibody was horseradish peroxidase-conjugated donkey anti-rabbit
IgG or sheep anti-mouse IgG (1:5000; Amersham Biosciences UK Limited, Little Chalfont
Buckinghamshire, UK). Horseradish peroxidase was detected using SuperSignal® West Femto
Maximum Sensitivity Substrate (Pierce Biotechnology Inc., Rockford, IL).
Determination of marker expression after immunohistochemistry
To calculate the percentage of cells expressing each marker in the Thy-1+ and Thy-1- cell
fractions, 1084 ± 72 immunohistochemical stained cells in 35 ± 3 separate microscopic fields
were counted for each analysis. Data were analyzed using SigmaStat 2.01 software (SPSS
Scientific, Erkrath, Germany) and are reported as the mean ± SEM.