X9090

To examine hepatic morphology and assess liver fibrosis, liver specimens were fixed 10% neutral buffered formalin, embedded in paraffin, and cut into 4 μm sections. Specimens were deparaffinized, hydrated, and stained as usual method with standard hematoxylin and eosin staining (H&E) and Sirius red staining as previously described^{52 (link)}. For immunohistochemistry (IHC), sections were incubated for 10 min in 3% hydrogen peroxide to block endogenous peroxidase. Antigen retrieval was performed by heating in 10 mM sodium citrate buffer (pH 6.0) for 10 min or incubating with 0.2% pepsin for 10 min. Sections were blocked in protein blocking solution (X9090; Dako, Carpinteria, CA, USA) for 30 min and incubated with primary antibodies, active Caspase-3 (AF835; R&D Systems, Minneapolis, MN, USA), F4/80 (ab6640; Abcam, Cambridge, MA, USA), Fpr2 (ab203129; Abcam), CD68 (ab125212;Abcam) or non-immune sera to demonstrate staining specificity at 4 °C overnight. Polymer horseradish peroxidase (HRP) anti-rabbit (K4003; Dako) or HRP anti-rat IgG (A110-105P; BETHYL, Montgomery, Texas, USA) was used as the secondary antibody. 3,3'-Diaminobenzidine (DAB) (K3466; Dako) was employed for the detection procedure.

For double immunofluorescence staining, cultured human pHSCs on coverslips or cytospun mouse pHSCs were fixed and permeabilized with 4% paraformaldehyde and Triton X-100, respectively. These cells were washed with TBS and blocked in protein blocking solution (X9090; Dako) for 30 min and incubated with first primary antibody, CD44ICD (KAL-KO601; CosmoBio; Carlsbad, CA, USA) or MMP14 (af918; R&D Systems) at 4 °C overnight and followed by Alexa Fluor 568-conjugated goat anti-rabbit IgG (A10042, Invitrogen) or Alexa Fluor 647-conjugated donkey anti-goat IgG (A21447, Invitrogen) for 30 min. For second primary antibodies, sections were incubated with α-SMA (A5228; Sigma-Aldrich) or CD44 (ab157107; Abcam) for 2 h at room temperature and followed by Alexa Fluor 488-conjugated chicken anti-mouse IgG (A21200, Invitrogen) or Alexa Fluor 568-conjugated goat anti-rabbit IgG (A10042, Invitrogen) for 30 min. 4’,6-diamidino-2-phenylindole (DAPI) were employed in the counterstaining procedure. The specimens were then observed and analyzed by confocal microscope DMi8-S (Leica Microsystems, Wetzlar, Germany) or CLSM21 (Carl Zeiss Inc., Thornwood, NY, USA).

To examine hepatic morphology and assess liver fibrosis, liver specimens were fixed 10% neutral buffered formalin, embedded in paraffin, and cut into 4 μm sections. Specimens were deparaffinized, hydrated, and stained as usual method with standard hematoxylin and eosin staining (H&E) and Sirius red staining as previously described [17 (link)]. For immunohistochemistry (IHC), sections were incubated for 10 min in 3% hydrogen peroxide to block endogenous peroxidase. Antigen retrieval was performed by heating in 10 mM sodium citrate buffer (pH 6.0) for 10 min or incubating with 0.2% pepsin for 10 min. Sections were blocked in protein blocking solution (X9090; Dako, Carpinteria, CA, USA) for 30 min and incubated with primary antibodies, rabbit anti-Col1a1 (NBP1-30054; Novus Biologicals, LLC, USA), mouse anti-α-SMA (A5228; Sigma-Aldrich), rat anti-F4/80 (ab6640; Abcam, Cambridge, MA, USA) and rabbit anti-CD68 (ab125212; Abcam) or non-immune sera to demonstrate staining specificity at 4 °C overnight. Polymer horseradish peroxidase (HRP) anti-rabbit (K4003; Dako), anti-mouse (K4001; Dako) or HRP anti-rat IgG (A110-105P; BETHYL, Montgomery, Texas, USA) was used as the secondary antibody. 3,3'-Diaminobenzidine (DAB) (K3466; Dako) was employed for the detection procedure.