3.1. Animal and CCl4 model
Six-week-old C57BL/6 mice (BioLASCO) were intraperitoneally injected with 0.5 µL/g mouse CCl4 (Alps Pharmaceutical Industry, 1/10 diluted in corn oil, WAKO) twice a week for six weeks to induce liver fibrosis (Taura et al., 2010).
Five mice were sacrificed every two weeks one day after the last injection and the livers were removed. The biggest lobe of mice liver were removed from the organ and equally dissected into three parts, one for total RNA extraction and the other two for cryosection and further analysis.
3.2. Whole liver RNA extraction
Liver tissue was homogenized in 3 mL Tripure (Roche) to extract total RNA.
Homogenized tissue was centrifuged at 12000g in 4℃ for 15 minutes to remove the pellet. 200 µL/mL Tripure chloroform (MP Biomedical) was added into the supernatant then the mixture was centrifuged at 12000g in 4℃ for 15 minutes.
The upper aqueous phase was transferred into a new microcentrifuge tube before 500 µL/mL Tripure isopropanol (Wako) was added and centrifuged at 12000g in 4
℃ for 15 minutes to precipitate the RNA. Precipitated RNA was washed twice
with 70% ethanol before air-dried and dissolved in 50 µL DEPC/ddH2O. The quality of RNA was determined by Nanodrop 2000 (Thermo) and electrophoresis
before reverse transcribed into cDNA.
3.3. Hematoxylin and eosin (H&E) staining
Liver tissue were fixed in 10% formalin (Sigma Aldrich, diluted in PBS [137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4]) for 10 minutes, embedded in paraffin and dissected. The sections were then deparaffinized and rehydrated before stained with hematoxylin. After washed by distilled water, the samples were immersed in eosin solution, washed again, dehydrated, cleared with xylene and mounted for image analysis.
3.4. Masson’s Trichrome staining
Liver tissue were fixed in 10% formalin for 10 minutes, embedded in paraffin and dissected. The sections were then deparaffinized and rehydrated before stained with Weigert's hematoxylin solution. After washed by distilled water, the samples were immersed in Biebrich scarlet-acid fuchsin solution, washed again, and immersed in phosphomolybdic-phosphotungstic acid solution to remove the red color. After washed, aniline blue solution was used to stain collagen in the tissue. Finally, the sections were decolorized in 1% acetic acid, washed, dehydrated, cleared with xylene and mounted for image analysis.
3.5. Liver immunofluorescence staining
Liver tissue was fixed in OCT (Sakura Finetek) and cryosectioned into 8 µm
thick sample using Leica CM 1900 (Leica). The cryostats were then fixed with 5% paraformaldehyde (Sigma Aldrich) for 15 minutes before permeabilized with 0.1% Triton X100 (Riedel-de Haën) for 5 minutes. 4% fetal bovine serum (FBS, diluted in PBS) were used as blocking buffer. After blocking for an hour in room temperature, the samples were incubated in primary antibody solution overnight in 4℃. Then the samples were incubated in secondary antibody at room
temperature for an hour, stained with Hoechst 33342 for 10 minutes and mounted before analyzed by IN Cell Analyzer (GE Healthcare). PBS served as wash buffer between each staining process. The antibodies used in this study and its dilution condition were shown in Table 1.
3.6. Laser microdissection (LMD) and RNA isolation
Liver tissue was fixed in OCT and cryosectioned into 8µm thick sample using Leica CM 1900 (Leica). The samples were then placed onto PET slide (Leica) for laser microdissection (LMD) using Leica LMD 7000 (Leica). Around 50mm2 of selected region were cut for 1 preparation of RNA. The RNA was isolated using High Pure FFPET RNA Isolation Kit (Roche). First the tissue pellet were digested in 100 µL RNA Tissue Lysis Buffer, 16 µL 10% SDS, and 40 µL Proteinase K for 30 minutes at 85℃, and further digested in 80 µL Proteinase K at 55℃ for another 30 minutes. Then 325 µL RNA Binding Buffer and 325 µL 100% EtOH
were added into the clear tissue lysate before the lysate were transferred onto the High Pure Collection Tube and centrifuged for 30 seconds at 6000g. The High Pure Collection Tube was centrifuged again for 2 minutes at 14000g to dry the filter. 100 µL DNase was added into the High Pure Collection Tube and incubated for 15 minutes at room temperature before 500 µL Wash Buffer I was added into the Collection Tube and centrifuged for 20 seconds at 6000g. Then the High Pure Collection Tube was washed twice by adding 500 µL Wash Buffer II and
centrifuge for 20 seconds at 6000g. Finally the High Pure Collection Tube was dried by centrifuged at 14000g for 2 minutes before 25 µL RNA Elution Buffer were added and centrifuged at 6000g for 1 minutes. The quality of RNA was determined by Nanodrop 2000 before reverse transcribed into cDNA.
3.7. Cell culture
Huh7 HCC cell line were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% FBS (GE Healthcare) and 1% penicillin/streptomycin/glutamine (PSG, Life Technologies) and maintained at 37℃ in a humidified incubator with 5% CO2. Hypoxic culture were conducted with same culture medium in incubator with 5% CO2 and 1% O2.
3.8. Cellular RNA extraction
The cells were scraped using Tripure by tips after the removal of culture
medium and washed once by PBS. 200 µL/mL Tripure chloroform was added into the supernatant then the mixture was centrifuged at 12000g in 4℃ for 15 minutes.
The upper aqueous phase was transferred into a new microcentrifuge tube before 500 µL/mL Tripure isopropanol was added and centrifuged at 12000g in 4℃ for 15 minutes to precipitate the RNA. Precipitated RNA was washed twice with 70%
ethanol before air-dried and dissolved in 50 µL DEPC/ddH2O. The quality of RNA was determined by Nanodrop 2000 and electrophoresis before reverse transcribed into cDNA.
3.9. Cellular protein extraction
The cells were scraped using RIPA buffer (50 mM Tris-HCl, 1% NP-40, 0.5%
Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, pH7.4) supplemented with cOmplete Cock Tail Protease Inhibitor (Roche) by tips after the removal of culture medium and washed by PBS. The total cell lysate were then centrifuged at 12000g in 4℃ for 10 minutes to remove the pellet. The concentration of total cell protein were determined by Bradford method (Bio-Rad Protein Assay) in ELISA reader (Thermo Multiskan FC).
3.10. Quantitative reverse transcription PCR (qRTPCR)
2 µg or 1 µg (LMD sample) total RNA was first reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied biosystem)
according to the manual using the following protocol: 25℃/10min→37℃/
120min→85℃/10min→4℃. qPCR was performed using iQ SYBR green
detection system in Bio-Rad CFX96 (Bio-Rad) using the following protocol: 95℃
/2min the℃/ 5sec→65℃/30sec]X39 cycles→cy℃/ 5sec→65℃/5secc→℃. The expression level of target genes was normalized to β-Actin and 18S rRNA (for Huh7 sample) or β-Actin and glyceraldehyde-3-phosphate dehydrogenase (GAP, for LMD sample) expression. The primers used for qPCR were shown in Table 2.
3.11. Western blotting
10 µg protein samples were mixed with 5x sample buffer (250 mM Tris-HCl, 10% SDS, 0.5% (w/v) bromophenol blue, 50% glycerol, 5% β-mercaptoethanol, pH6.8) and placed on dry bath (Major Science Dry Bath Incubator) under 100℃
for 10 minutes. SDS-polyacrylamide gel (10% separation gel and 4% stacking gel) and TGS buffer system (50 mM Tris-HCl, 380 mM Glycine, 0.1% SDS, pH8.3) were used to separate total proteins. Electrophoresis were performed according to the following protocol: 100V for 20 minutes and 150V for 1.5 hour. Then the proteins were transferred onto pre-rinsed PVDF membrane (Roche) in western transfer buffer (25 mM Tris-HCl, 192 mM Glycine, pH 8.3) with Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad) for 1 hour. The current used for transfer was 1.5 mA/cm2. The membrane was washed with TBST (10 mM
Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH8.0) at least twice between each step.
4% skim milk (diluted in TBST) was used for blocking process, and the time duration was an hour at room temperature. Primary antibody solution (diluted in gelatin-NET [0.25% gelatin, 0.15 M NaCl, 5 mM EDTA, 0.05% Tween-20, 50 mM Tris, pH8.0] or 4% skim milk) were added after blocking and following wash and incubated in 4℃ overnight. Then the membrane was washed thrice before replaced into second antibody solution and incubate for 1 hour. At last the membrane was washed twice with TBST and once with ddH2O before rinsed in HRP substrate (WBKLS0500, Millipore) for 1 minutes and analyzed by
BioSpectrum 2D Imaging System (UVP BioSpectrum 800). The antibodies used in this study and its dilution condition were shown in Table 1.
3.12. Flow cytometry
For surface marker analysis, cells were harvested with Trypsin solution (Life Technologies), recovered and washed in culture medium before shaked in FcR blocking solution (Miltenyi Biotec) for 20 minutes at 4℃. Primary conjugated antibody (or isotype control) were added into each samples and shaked for an hour at 4℃. Cells were then stained with propidium iodide (PI, Life Technologies) for 10 minutes before washed with PBS and resuspended with 3% FBS (diluted in PBS). Cell analyzer (BD FACSCanto II) was used to analyze the expression of
markers. The antibodies used in this study and its dilution condition were shown in Table 1.
For intracellular antigen analysis, cells were first harvested with Trypsin solution (Life Technologies), fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.5% Tween-20 (Shimakyu's pure chemical) for another 15 minutes. Then the cells were blocked with 4% FBS for an hour, incubated in primary antibody solution for an hour, and finally mixed with secondary antibody solution for another hour. After stained with PI for 10 minutes and resuspended with PBS, cells were analyzed using cell analyzer. The antibodies used in this study and its dilution condition were shown in Table 1.
3.13. Cell immunofluorescence staining
After the removal of culture medium, Huh7 were first fixed with 5%
paraformaldehyde for 7 minutes then permeabilized with 0.1% Triton X100 for 15 minutes. 4% FBS (diluted in PBS) were used as blocking buffer. After blocking for an hour in room temperature, the samples were incubated in primary antibody solution overnight in 4℃. Then the samples were incubated in secondary antibody at room temperature for an hour, stained with Hoechst 33342 for 10 minutes before analyzed by IN Cell Analyzer (GE Healthcare). The cells were washed by PBS between each staining steps. The antibodies used in this study and its dilution
condition were shown in Table 1.
3.14. Image processing
The histochemical staining images were taken by Dr. Hsiao-Mei Chao of Taipei Medical University Hospital. All immunofluorescence images were first obtained by IN Cell Analyzer (GE Healthcare), and the local background were subtracted before merging channels and cropped by ImageJ. Results of flow cytometry were analyzed using FlowJo software and displayed in overlayed histogram.
3.15. Statistics
qRTPCR data in the bar charts represent means ± SEM and were obtained from average data of three independent experiments. Statistical significance was calculated using a two-tailed Student’s t-test. Differences with a P value of less than 0.05 were considered significant, and a P value of less than 0.01 were considered really significant.