Ab180904

The primary antibody used for immunostaining was an Anti-interleukin-17A-receptor antibody (ab180904, Abcam) diluted at 1:50. This step was followed by a secondary antibody conjugated to FITC (ZF-0311, ZSGB-BIO) diluted at 1:250. Samples were examined with fluorescent microscopy (Leica).

Slides of primary lung fibroblasts were prepared and fixed with 40 g/L paraformaldehyde at room temperature for 15 min, washed three times with PBS, and blocked with 10% goat serum. The sections were incubated with primary anti-rabbit antibodies against α-SMA (ab5831, 1:500, Abcam, UK), HDAC3 (ab7030, 1:500, Abcam, UK), and IL17RA (ab180904, 1:500, Abcam, UK) overnight at 4°C. Then, the sections were incubated with secondary antibodies against Alexa Fluor® 647-labeled fluorescence goat-anti-rabbit antibody against immunoglobin G (IgG) H and L (ab150079, 1:1,000, Abcam, Cambridge, UK) and FITC-labeled fluorescence goat-anti-mouse antibody against IgG H and L (ab6785, 1:1,000, Abcam, Cambridge, UK) for 45 min. Following three PBS washes, the sections were sealed with anti-fluorescent quenching mounting medium Vectashield (Vector Laboratories Inc., Burlingame, CA), and observed and photographed under a fluorescence microscope.

Human lung tissues from controls, patients with IPF, and patients with RA-ILD or mouse lung fibroblasts were lysed by lysis buffer (C0481, Sigma-Aldrich Chemical Company, St. Louis, MO, United States) to extract total protein. Protein loading buffer was added to the supernatant. After boiling for 5 min, 20 μg of protein sample was electroporated onto a polyvinylidene fluoride membrane by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Millipore, Billerica, MA, United States). The membrane was added with Tris-buffered saline containing Tween 20 (TBST) diluted primary antibodies IL17RA (ab180904, 1:1,000, Abcam), HDAC3 (ab219376, 1:1,000, Abcam), and GAPDH rabbit anti (ab181602, 1:10,000, Abcam) for incubation overnight at 4°C. HRP-labeled secondary antibody (ab99702, 1:1,000, Abcam) was added and incubated for 1 h. Enhanced chemiluminescence (Shanghai Baoman Biotechnology Co., Ltd., Shanghai, China) was used for developing. GAPDH was used as an internal reference. The Image J was used to analyze the luminosity of each band, and the ratio of the value of the target protein to the internal reference was calculated.

For immunohistochemistry, the slides of cells were incubated with primary rabbit anti-mouse antibody against IL-17R (1:50, ab180904; Abcam, Cambridge, UK) at 4 °C overnight, and then were incubated with horseradish peroxidase conjugated goat anti-rabbit IgG antibody (PV-9000, Zisbio, Beijing, China) at 37 °C for 30 min. After rinsing with PBS for three times, 3′3-diaminobenzidine-tetrahydrochloride was applied on the slides as a chromogen for 1–5 min. Slides were counterstained in haematoxylin for 5–10 min.
For immunofluorescence, the slides of cells were incubated with primary mouse anti-mouse antibody against Cytokeratin18 (1:50, ab668; Abcam, Cambridge, UK), mouse anti-mouse antibody against E-cadherin (1:50, ab76055; Abcam, Cambridge, UK), or rabbit anti-mouse antibody against Vimentin (1:50, ab92547; Abcam, Cambridge, UK) at 4 °C overnight, and then were incubated with Alexa Fluor 594-conjugated Goat Anti-Mouse IgG(H + L)(SA00006-3, Proteintech, IL, USA) or Alexa Fluor 594-conjugated Goat Anti-Rabbit IgG(H + L)(SA00006-4, Proteintech, IL, USA). Finally, slides were stained with DAPI (4′,6-diamidino-2-phenylindole) at 37 °C for 10 min.

To determine IL-17RA expression in the PVN, rats were transcardially perfused with 4% paraformaldehyde. Brains were removed, post-fixed in 4% paraformaldehyde for 24 h at 4°C and then cryoprotected in 30% sucrose for 48 h at 4°C. Brains were sectioned into 20 μm coronal sections using a cryostat. The sections were incubated with a rabbit anti-rat primary antibody to IL-17RA (ab180904, 1:50, Abcam, Cambridge, MA) and a mouse anti-rat primary antibody to NeuN (MAB377, 1:200, Millipore, Billerica, MA), followed by secondary antibodies Alexa fluor 488 goat anti-rabbit IgG (ab150077, 1:200, Abcam, Cambridge, MA) and Alexa fluor 568 goat anti-mouse IgG (ab 175702, 1:200, Abcam), respectively. To verify the transfection potential of the siRNAs by visualization of GFP in the PVN, rats were sacrificed without perfusion and brains were removed quickly. Brains were immediately frozen in liquid nitrogen and then sectioned into 20 μm coronal sections using a cryostat. Immunofluorescence for IL-17RA and NeuN and GFP were visualized by a confocal laser-scanning microscope (Zeiss LSM 710, Carl Zeiss, Inc., Oberkochen, Germany).

In parallel to the investigation of cell polarization, the gene and protein levels of RANKL in the Th cells (LPS group, LPS + DC group and LPS + DC + Cal group) following a 5‐day incubation were determined by qRT‐PCR and Western blot analysis (for details, see Sections 2.8 and 2.9). In addition, immunofluorescence and flow cytometry assays were performed to assess the proportion of IL‐17⁺/RANKL⁺ cells.¹³ Briefly, after Th cell smears were prepared, Th cells were incubated with primary antibodies against RANKL (ab45039; Abcam) and IL‐17 (ab180904; Abcam) overnight at 4°C and then incubated with secondary antibodies (Alexa Fluor 488‐conjugated goat anti‐rabbit and Alexa Fluor 647‐conjugated goat anti‐mouse) for 50 minutes in the dark. Finally, the cells were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and then visualized and imaged with a fluorescence microscope (NIKON ECLIPSE C1, Nikon DS‐U3). Similarly, for detecting the proportion of IL‐17⁺/RANKL⁺ cells by flow cytometry, cells were immunostained with PE‐conjugated anti‐mouse RANKL (IK22/5, BioLegend) and APC‐conjugated anti‐mouse IL‐17 antibodies, and the immunostained IL‐17⁺/RANKL⁺ cells were detected and analysed with a Beckman Coulter Epics XL flow cytometer (Beckman Coulter).³²

The brain tissue and colon were removed, fixed, and dehydrated to further process for immunofluorescence. After blocking for 1 h at room temperature, brain sections were incubated with primary antibodies overnight at 4 °C. The primary antibodies used in this study were tyrosine hydroxylase (TH) (MAB318, Millipore, Bedford, MA, USA), allograft inflammatory factor 1 (Iba1) IgG (019-19741, Wako, Japan), cleaved active caspase-3 (9661, Cell Signaling Technology, Danvers, MA, USA), LRRK2 (MJFF2 [c41-2]) (ab133474, Abcam, Cambridge, UK), MAP2 lgG (ab32454, Abcam), Iba1 lgG (MA5-27726, Thermo Fisher Scientific, Waltham, MA, USA), and IL-17RA lgG (ab180904, Abcam). Subsequently, the sections were incubated with Alexa 488- or 555-conjugated secondary antibodies (4408, 4413, Cell Signaling Technology) for another 1 h at room temperature. Finally, sections were viewed under a Nikon microscope (Japan). The number and density of cells were measured in a double-bind manner with ImageJ v1.51 software and two individuals. For TH+ cell counting, five consecutive sections containing the substantia nigra were selected per animals.