Histovt one

For the immunohistochemical (IHC) analysis of lung tissues, paraffin-embedded sections were deparaffinized and subsequently applied to heat-induced antigen retrieval in HistoVT One (Nacalai Tesque). The sections were incubated with rabbit anti collagen IV antibody (Abcam). Protein binding was detected with a Vectastain elite ABC kit (Vector Laboratories Ltd, Peterborough, UK) and 3,3’-diaminobenzidine, tetrahydrochloride (DAB) as a substrate, and counterstained with hematoxylin.

In vitro bromodeoxyuridine (BrdU) incorporation was performed to analyze cell proliferation. Human VSMCs were plated in a Nunc^® Lab‐Tek^® II CC2™ Chamber Slide™ (Sigma‐Aldrich) at a density of 1.0 × 10⁴ cells/cm² at 2 days after transduction of indicated lentiviral vectors. The lentivirus‐containing medium was diluted twice for overexpression experiments due to high cell toxicity compared with the knockdown experiments. Another 2 days later, cells were stimulated with 10 μM BrdU (Sigma, B9285) for 4 h and fixed with 4% paraformaldehyde (PFA) for 30 min at 4°C. The slides were autoclaved (90°C, 20 min) in HistoVT One (Nacalai Tesque) for heat‐mediated antigen retrieval. Then, cells were permeabilized in 1.0% Triton for 15 min and blocked in 5% donkey serum/PBS for 15 min, followed by incubation with an anti‐BrdU antibody (Abcam, ab6326, 1:250 dilution) at 4°C overnight. The slides were rinsed three times and then incubated with donkey anti‐rat IgG secondary antibody Alexa Fluor 594 (1:200; Thermo Fisher A11007) with DAPI (0.5 μg/ml) at room temperature for 1 h. The slides were washed and mounted in VECTASHIELD^® Mounting Medium (Vector Laboratories, H‐1000) and then observed using an Axio Observer 7 (Zeiss) with a 10× objective. Images were randomly acquired, and the percentage of BrdU‐positive cells in each image was counted using ImageJ64 for analysis.

PIT model mice, at 24 h after stroke onset, were perfused intracardially with PBS followed by 4% paraformaldehyde (PFA) in PBS. The entire brain was removed and immersed in 800 μl of 4% PFA at 4 °C for 24 h. After dehydration in order using 10%, 20%, and 30% sucrose in 0.1 m PBS, brain tissues were embedded into O.C.T. compound (Sakura FineTek, Tokyo, Japan) and frozen at −80 °C. Thereafter, 25-μm-thin sections were prepared using Cryostat (CM3050S; Leica, Wetzlar Germany). The tissue section was fixed again with 4% PFA, washed with PBS, and immersed in Histo VT one (Nacalai Tesque, Tokyo, Japan) at 70 °C for 20 min, followed by incubation with 0.2% Triton X-100 in PBS at room temperature for 10 min. After washing with 0.5% Tween 20 in PBS, tissue sections were blocked with 3% BSA and 0.5% Tween 20 in PBS. Primary antibodies of HPSE (H-80) and secondary antibodies used in this study are shown in Table S2. Tissue sections were observed using a confocal microscope LSM 780 (Carl Zeiss, Oberkochen, Germany).

The primary antibodies used were antihuman SR-B1 rabbit polyclonal antibody (#5193; ProSci, Poway, CA, USA), antihuman LOX-1 rabbit polyclonal antibody (#11837-1-AP; Proteintech, Rosemont, IL, USA), and antihuman CD68 mouse monoclonal antibody (#M0814; Dako, Glostrup, Denmark). Immunostaining was carried out on 5-µm-thick cryostat sections. For antigen retrieval, sections were immersed in Histo VT One (Nacalai Tesque, Kyoto, Japan) at 70 °C for 40 min. The slides were then incubated overnight at 4 °C with the primary antibodies. Color development was achieved using the streptavidin–biotin amplification technique (ChemMate EnVision kit; Dako). Peroxidase activity was visualized by diaminobenzidine solution. Sections were counterstained with hematoxylin. Control specimens developed without the primary antibody were used to verify that nonspecific binding was not detectable. Consecutive sections were stained with hematoxylin and eosin (HE) for the assessment of mucosal pathology and the degree of eosinophil infiltration.
All procedures contributing to this work complied with the ethical standards and with the Helsinki Declaration. The study protocol was approved by the Institutional Review Board at the Hiroshima University School of Medicine on 11 June 2018 (Approval No. Hi-136-2). Written informed consent was obtained from all patients prior to their participation.

Paraffinized sections of synovium from RA and OA patients were deparaffinized and rehydrated by successive washes with xylene and graded ethanol. Antigens were retrieved with HistoVT One (Nacalai Tesque, Inc., Tokyo, Japan). Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol. The slides were then incubated with normal serum for 1hour at room temperature and then with the primary anti-IEX-1 antibody (Sigma-Aldrich) or with rabbit polyclonal IgG (Abcam) overnight at 4°C. The slides were incubated with a biotinylated secondary antibody for 30 min, followed by avidin and biotinylated horseradish peroxidase (Santa Cruz Biotechnology, Inc., CA, USA) for immunohistochemical staining.

For immunostaining, cultured slices were fixed in 4% PFA at 4°C for 24 hours. The fixed samples were rinsed 3 times with PBS and heated in 10% HistoVT One (06380‐05; Nacalai Tesque) for 20 minutes at 90°C. The slices were then permeabilized and blocked for 1 hour at 4°C in PBS + 0.3% Triton X‐100 with 10% goat serum. The samples were subsequently incubated with primary antibodies in PBS + 0.3% Triton X‐100 with 10% goat serum at 4°C for 48 hours with agitation. The samples were rinsed three times with PBS and then incubated with secondary antibodies in PBS + 0.3% Triton X‐100 with 10% goat serum at 4°C overnight with agitation. The samples were rinsed 3 times with PBS. To label the nuclei, 0.1% Hoechst was added to PBS during the second rinse. After the rinse, the samples were embedded in Permafluor (Thermo Fisher, Waltham, MA, USA). The following primary antibodies were used for immunostaining: mouse anti‐NeuN (1:1000; MAB377; Merck Millipore) and guinea pig anti‐Iba1 (1:500; 324 006, Synaptic System, Goettingen, Land Niedersachsen, Germany). The following secondary antibodies were used for immunostaining: Alexa Fluor 594‐ and 647‐conjugated secondary antibodies (1:500; Thermo Fisher).