Immunohistochemistry
Paraffin-embedded liver samples were cut into approximately 4 μm sections and incubated for 6 min with proteinase K (Agilent tech. Inc., Santa Clara, CA) or autoclave-heated for 10 min in 0.01 M citrate buffer for antigen retrieval. Sections were washed thrice in PBS and blocked by incubation with 3% BSA in PBS for 30 min or with a blocking reagent (Cat #414321; Nichirei Bioscience Inc., Tokyo, Japan) for 60 min at room temperature. The expression levels of αSMA, F4/80, and Lymphocyte antigen 6 complex, locus G (Ly-6G) were determined using anti-αSMA (Agilent tech. Inc., Cat# MO851, RRID: not yet assigned), anti-F4/80 (Bio-Rad Laboratories Inc., Cat# MCA497R, RRID: AB_323279), and anti-Ly-6G (BioLegend Inc., San Diego, CA, Cat# 127602, RRID: AB_1089180) antibodies, respectively. After washing, the sections used for F4/80 and Ly-6G detection were incubated with biotinylated anti-rat IgG (Vector laboratories Inc., Newark, CA, Cat# BA-9400, RRID: AB_2336202) and analysed based in the formation of a streptavidin-biotin complex (Vector lab. Inc., Cat# PK-4000) with 3,3’-diaminobenzidine tetrahydrochloride (DAB-4HCl) solution (Agilent tech. Inc., Cat# K3468). Sections used for αSMA detection were incubated with Simple stain MAX PO (Nichirei Bioscience Inc., Cat# 414321) and with detection using DAB-4HCL solution. The sections were counterstained with haematoxylin. The numbers of F4/80- or Ly-6G- positive cells per ×40 high-power field were counted manually. Ten high-power fields were examined per mouse.
The expression of C-type lectin domain family 4 member F (CLEC4F), Glycoprotein nonmetastatic melanoma protein B (GPNMB), CC chemokine receptor (CCR)2, Sialophorin (SPN), CCR1, and CCR5 was determined using Ventana discovery ultra (Roche Diagnostics K.K., Basel, Switzerland) according to Ruo discovery universal (ver. 0.00.0394) staining. Briefly, for immunohistochemistry, antigen retrieval was performed using CC1 buffer (Roche Diag. K.K., Cat# 518-108939) for 64 min at 95 °C. The sections were then incubated for 4 min at 37 °C with Inhibitor CM, which was included in the Chromomap kit (Roche Diag. K.K., Cat# 518-100803). The sections were stained for 32 min at 37 °C with the respective primary antibody, against CCR1 (Abcam Inc., Cat# ab140756, RRID: none yet assigned) and CCR5 (Bioss Inc., Woburn, MA, Cat# bs-2514R, RRID: AB_10857802). They were then incubated for 16 min at 37 °C with the secondary antibody, Omnimap anti-rabbit HRP (Roche Diag. K.K., Cat# 518-102135). Next, the sections were incubated for 12 min at 37 °C with Discovery DAB CM and Discovery H2O2 CM and for 4 min at 37 °C with Discovery Copper CM, all of which were included in the Chromomap kit (Roche Diag. K.K., Cat# 518-100803) to detect the signal. The sections were counterstained with haematoxylin.
For immunofluorescence, antigen retrieval and inhibition of endogenous peroxidase activity were performed in the same manner as for immunohistochemistry. Sections for CLEC4F detection were blocked with Goat F(ab) anti-mouse IgG H&L (Abcam Inc., Cat# ab6668) for 16 min at 37 °C. Sections were stained for 32 min at 37 ºC with the respective primary antibody against GPNMB (Abcam Inc., Cat# ab188222, RRID: none yet assigned), CCR2 (Abcam Inc., Cat# ab273050, RRID: AB_2893307), CLEC4F (R&D systems Inc., Cat# MAB2784, RRID: AB_2081338), and SPN (Thermo Fisher Scientific Inc., Cat# PA5-96540, RRID:AB_2808342). They were then incubated for 16 min at 37 ºC with the secondary antibody, Omnimap anti-rabbit HRP (Roche Diag. K.K., Cat# 518-102135) for anti-GPNMB, anti-CCR2 and anti-SPN primary antibodies or with Omnimap anti-rat HRP (Roche Diag. K.K., Cat# 518-110932) for anti-CLEC4F primary antibody. The sections were then incubated for 32 min at 37 ºC with Discovery Cy5 (Roche Diag. K.K., Cat# 518-113667) for signal detection.
For multiple staining, the processes of antigen retrieval, inhibition of endogenous peroxidase activity, and reaction with primary and secondary antibodies were conducted in same as for the respective immunohistochemistry or immunofluorescence process, except an incubation step for 4–8 min at 95 ºC was added between each staining step to denature the residual antibodies. For signal detection, the sections were incubated for 32 min at 37 ºC with Discovery Cy5 (Roche Diag. K.K., Cat# 518-113667), Discovery rhodamine (Roche Diag. K.K., Cat# 518-111861), Discovery FITC (Roche Diag. K.K., Cat# 518-111878) and Discovery DCC (Roche Diag. K.K., Cat# 518-113988) at the end of each staining step. Positive areas in the sections were quantified using a BZ-X810 microscope and Hybrid cell count software (Keyence Corp, Osaka, Japan) as previously described (Yoshimoto & Shinya, 2022).