Goat serum

Cells were grown on 24 mm round coverslips (Paul Marienfeld). After 2 hours of serum starvation and pretreatment with or without 5 μg/ml euphol, cells were stimulated with TGF-β(20 pM) for 30 min. Cells were washed with phosphate buffered saline (PBS) and fixed in cold methanol for 10 min. After washings with PBS, cells were blocked with 5% goat serum (Dako) in 1% BSA/PBS. After incubation with mouse-anti-Smad2 (H-2; Santa Cruz Biotechnology) at 1:100 dilution in 1% BSA/PBS for 18 hours at 4°C, cells were incubated with donkey anti-mouse FITC (Gene Tex) at RT for 1 h. Coverslips were mounted with slow fade gold anti-fade reagent and DAPI (Invitrogen). Photomicrographs were taken with a Zeiss Axio Observer Z1 microscope equipped with a Photometrics HQ2 camera.

After washing with PBS, N2a cells were fixed for 30 min with 4% formaldehyde (Polysciences, PA, USA) in PBS at room temperature. Cells were washed three times with PBS, followed by permeabilization with 0.1% Triton-X100 (Acros) in PBS for 10 min. After blocking for 20 min with 5% goat serum (Dako) and 1% BSA (Sigma-Aldrich) in PBS (blocking buffer), cells were incubated with the primary homemade antibody for endogenous NBEA (1/1,000), diluted in blocking buffer, for 1h. After three washes, cells were incubated with a goat secondary Alexa594- or Alexa488-conjugated antibody (Life technologies) and DAPI (1/10,000) for 1h. Finally the cells were washed and mounted on microscope slides using Vectashield mounting medium (Vector laboratories, Canada). Cells were analyzed using the Olympus Fluoview FV1000 confocal laser scanning microscope and a 63x immersion oil objective.
The tool ‘Z project’ in ImageJ software was used to make a 2D Z-projection of a confocal Z-stack. All confocal images shown are single confocal planes, unless otherwise specified.
Quantification of nuclear EGFP fluorescence intensity was performed using Cell Profiler cell analysis software [39 (link)]. At least 300 transfected cells were measured per condition.

Fixed cells were washed three times with 1x PBS, permeabilized with 0.5% Triton X-100 PBS for 5 min at room temperature, and blocked by using 5% goat serum (Sigma) in 0.5% Tween PBS for 20 minutes at room temperature. Incubation with a mouse monoclonal IgG2a anti-DNA/histone (Millipore, Billerica, Massachusetts, USA) and a rabbit anti-human myeloperoxidase (Dako) in 5% goat serum 0.5% Tween PBS was carried out for 1h at room temperature. Isotype controls, murine myeloma IgG2a and IgG from rabbit serum (both from Sigma) were included to avoid unspecific signals. Samples were later washed with PBS three times and subsequently incubated with an Alexa-Fluor-488-labelled goat-anti-mouse IgG2a antibody, and an Alexa-Fluor-633-labelled goat-anti-rabbit IgG, for 45 min at room temperature in the dark. After washing, slides were mounted in ProlongGold^® antifade with DAPI (Invitrogen), and cover slips were surrounded with clear nail polish and stored at 4°C in the dark until confocal fluorescence microscopy analysis.

Resected lungs were fixed in 10% formalin, processed and embedded in paraffin blocks, and 3 µm thick sections were processed on positively charged slides. After deparaffinization by serial incubations in xylene and ethanol (100%, 95%, and 70%), antigen retrieval was carried out by incubating the slides in 1 mM ethylenediaminetetraacetic acid (EDTA) pH 9.0 (Sigma-Aldrich) in a water bath at 95 °C for 30 min. After washing with PBS (Sigma-Aldrich, Germany), endogenous peroxidase activity was blocked using 2% hydrogen peroxide (H₂O₂) for 10 min. The sections were then washed with PBS, permeabilized in 0.05% Triton X-100 for 10 min, blocked in 5% goat serum (Dako, 5301 Stevens Creek Blvd, Santa Clara, CA 95051, USA) for 30 min, and then incubated with primary antibody (Anti-GFP antibody (#2956 cell signaling technology) at 4 °C overnight in a humidified chamber. The slides were then washed in PBS and incubated with the EnVision™ + Dual Link System-HRP (Dako, USA) labeled secondary antibody for one hour at room temperature, followed by incubation with substrate chromogen solution (DAB chromogen (Dako). The sections were counterstained using hematoxylin solution, dehydrated, mounted using DPX (Sigma, USA) and visualized using a (NikonH600L) light microscope.

Frozen synovial tissue sections were fixed in acetone and blocked with 10% goat serum (Dako), followed by incubation with the Biotin blocking system (Dako). Stainings with mAb directed against TNF (clone 52B83; Hycult Biotech), CD45 (clone HI30; BioLegend), CD55 (clone JS11; BioLegend), CD68 (clone Y1/82A; BioLegend), CD90 (clone 5E10; BioLegend), CD163 (clone GHI/61; BioLegend), and vimentin (clone D21H3; Cell Signaling Technology) were performed overnight at 4°C, followed by incubation with Alexa Fluor 488/Alexa Fluor 555–conjugated goat anti-mouse and goat anti-rabbit secondary antibodies. Slides were mounted with Vectashield containing DAPI (Vector Laboratories) and analyzed on a fluorescence imaging microscope (Leica DMRA) coupled to a charge-coupled device camera, with results analyzed using Image-Pro Plus software (Media Cybernetics, Dutch Vision Components).

Cells were fixed in 4% (wt/vol) paraformaldehyde for 10 min, permeabilized with 0.2% Triton X‐100 for 5 min and blocked in 3% (vol/vol) goat serum (Dako) or donkey serum (Sigma) for 45 min. They were then incubated in primary antibodies for 45 min followed by secondary antibodies for 30 min (Alexa Fluor dyes, 1:1000, Invitrogen). All antibodies were diluted in the blocking buffer. Nuclei were counterstained with DAPI (Sigma) for 5 min and coverslips were mounted on slides with FluorSave (Merck). All procedures were performed at room temperature. Primary antibodies used in this study were Vimentin (1:100, Millipore), NFIA (1:250, abcam), GFAP (1:500, Dako), GFAP (1:500, Sigma), S100B (1:500, Dako), βIII‐tubulin (1:1000, Sigma), TDP‐43 (1:250, Abnova), NANOG (1:250, R&D Systems), SOX2 (1:250, Millipore), TRA‐1‐60 (1:250, Santa Cruz), OCT3/4 (1:250, Santa Cruz), SOX1 (1:100, R&D Systems), Nestin (1:1000, Millipore), Brachyury (1:100, R&D Systems), EOMES (1:600, abcam), FOXA2 (1:100, R&D Systems), GATA‐4 (1:100, Santa Cruz), SMI32 (1:250, Covance), and Caspase‐3 (1:500, Abcam).
Fluorescent imaging was performed on fields of view containing uniform DAPI staining using either an Axio Observer.Z1 (Zeiss) epifluorescence microscope or an LSM710 confocal microscope (Carl Zeiss). Images were processed and blindly analyzed by using the ImageJ64 (v 1.47) software.

Morphology was examined in tissue sections (5 μm) stained with H&E. The expression of FN, calpain-2, E-cadherin, and vimentin was analyzed using a two-step Immunohistochemical Stain Detection System (PV-9000, Zhongshan Golden Bridge Biotechnology) according to the manufacturer’s instructions. Briefly, the sections were deparaffinized, and antigen retrieval was achieved by microwave heating in 0.01 M citrate buffer for 20 min. The sections were sequentially incubated with 3% H₂O₂, blocked with goat serum (Dako) for 30 min, and incubated with the following primary antibodies at 4 °C overnight: FN (1:300), E-cadherin (1:250), calpain-2 (1:250), and vimentin (1:350). The sections were successively incubated with biotinylated secondary antibodies for 20 min and streptavidin-HRP for 2 min, and then stained with DAB substrate and counterstained with hematoxylin. Results were captured using a Leica DMI8 microscope and Leica X software (Leica) at ×400 magnification.