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).