Tenascin

Complementary mouse models of fibrosis were employed to evaluate the role of tenascin-C in vivo. First, 8-week-old female TNC^−/− mice (C57BL/6 N-TgH, from RIKEN, Japan) and C57BL/6J mice (The Jackson Laboratory) in parallel received bleomycin (10 mg kg⁻¹ per day) or PBS daily via s.c. injections for 10 days, and killed at various time points for up to 40 days after the last injection. All treatment groups consisted of at least five mice and experiments were repeated three times with consistent results. Cultures of fibroblasts were established by explantation from dorsal skin from wild-type and TNC^−/− mice. In a complementary non-inflammatory model of fibrosis, Tsk/+ mice (C57BL/6 background, The Jackson Laboratory) at 8 weeks of age were crossed with TNC^−/− mice to generate TNC^−/−;Tsk/+ mice. At 12 weeks of age, female TNC^−/−;Tsk/+ mice and C57BL/6J control mice were killed, and lesional skin and lungs were collected for analysis21 (link).
Four-micometre-thick sections of paraffin-embedded tissues were stained with haematoxylin and eosin or Trichrome. Thickness of the dermis and hypodermis, defined as the distance from the epidermal–dermal junctions to the dermal–adipose junction or to the loose connective tissue subjacent to the panniculus carnosus, respectively, were determined at five randomly selected sites per h.p.f. Sections of lungs stained with haematoxylin and eosin were scored for fibrosis63 (link) in a blinded manner by an expert pulmonary pathologist (K.R.). For immunofluorescence analyses, paraffin-embedded skin and lung sections were incubated with primary rabbit antibodies against αSMA (1:100), tenascin-C (T2H5, 1:500), LOX (100) or phospho-Smad2 (Cell Signaling, 1:100), followed by Alexa-fluor-labelled rabbit secondary antibodies. Nuclei were detected using DAPI. Slides were evaluated under a Zeiss UV Meta 510 confocal microscope. For immunohistochemistry, sections of paraffin-embedded skin and lungs were immunolabelled with primary rabbit antibodies against F4/80 (1:500, eBioscience, San Diego, CA) and CD3 (1:3000, Abcam), followed by appropriate biotinylated secondary antibodies (1:250, all from Jackson Immunoresearch, West Grove, PA) and detected using biotin complex conjugated with horseradish peroxidase (Vector Laboratories, Burlingame, CA) and DAB for colour development (Dako, Carpinteria, CA). Collagen content of the lungs was determined by hydroxyproline assays (Colorimetric Assay Kits, Biovision, Milpitas, CA).

Hematoxylin and eosin (H&E) stained sections of BM were examined, and representative tissue blocks were selected for further staining. Tissue sections (3 μm thick) were subjected to IHC using a Bond-max autostainer (Leica Microsystems, Buffalo Grove, IL, USA), as previously described 23 (link). The following antibodies were used: α-SMA (1:100 dilution; catalogue no. ab5694; Abcam, Cambridge, UK), FAP-α (1:1000 dilution; catalogue no. AF3715; R&D, Minneapolis, USA), S100A4/FSP1 (1:1000 dilution; catalogue no. HPA007973; Atlas, Bromma, Sweden), PDGFR-α (1:100 dilution; catalogue no. sc398206; Santa Cruz, Texas, USA), PDGFR-β (1:400 dilution; catalogue no. ab32570; Abcam), collagen type I (1:300 dilution; catalogue no. ab34710, Abcam), NG2 (1:600 dilution; catalogue no. ab139406; Abcam), Tenascin-C (1: 50 dilution; catalogue no. sc25328; Santa Cruz), and Twist1 (1:100 dilution; catalogue no. ab50887; Abcam). IHC slides were assessed by experienced pathologists (SSK and KHL), who were blinded to the clinical details. The staining intensity in stromal CAFs was initially graded from 0 through 3 and then grouped into two categories. The scores of immunostaining in stromal CAFs was given according to the relative ratio of the area stained by the CAF markers to the area of the tumor cells: 0, no staining; 1, ≤ 3%; 2, ≤ 10%; and 3 > 10%. Low expression was graded with 0 or 1, while high expression was graded with 2 or 3 for α-SMA, FAP-α, S100A4/FSP1, PDGFR-β, collagen type I, NG2, and Tenascin-C. Twist1 expression was defined as positive/high when nuclear staining was present, and as negative/low when nuclear staining was absent.

Cryosections were labelled by indirect immunofluorescence using single- and dual-label approaches. A minimum of five separate corneas were examined for each embryonic age studied. For single labels, sections were rehydrated with PBS containing 0.1% Tween20 (Sigma-Aldrich, Poole, UK; PBST) and incubated with primary antibodies overnight at 4 °C. These were the mouse monoclonals B3D6 to chicken fibronectin, M1B4 to chicken tenascin-C and 5C9 to chicken perlecan (all at 5 µg/mL in PBS) [67 (link),68 (link),69 (link)]. Sections were washed in PBST (three changes over 15 min) and then incubated with horse anti-mouse IgG conjugated with Dylight 488 secondary antibody for 6 h (Vector Laboratories, Peterborough, UK; 5 µg/mL). Sections were washed in PBST as above and mounted in Fluoroshield antifade mountant containing DAPI as a nuclear counterstain (Sigma-Aldrich, Poole, UK). Antibodies B3D6, M1B4 and 5C9 were obtained from the Developmental Studies Hybridoma Bank created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242, USA.
Dual labels for fibronectin and tenascin-C were possible due to the different IgG subtypes of the two primary antibodies—B3D6 is IgG2a and M1B4 is IgG1. Sections were incubated with a mixture of these two antibodies (5 µg/mL each) as above, washed and then incubated with a mixture of two isotype specific secondary antibodies: goat anti-mouse IgG2a-Alexa 488 conjugate and goat anti-mouse IgG1-Alexa 594 conjugate (5 µg/mL each; Invitrogen, ThermoFisher Scientific, Paisley, UK) and mounted as described above. Dual labels for fibronectin and type IV collagen used antibody B3D6 as above, mixed with polyclonal rabbit anti-chicken type IV collagen (5 µg/mL; anti-COL4A1 antibody, Antibodies-online, Aachen, Germany), using the same incubation times and procedures as described above. The secondary antibodies were horse anti-mouse IgG-Dylight 488 conjugate and horse anti-rabbit IgG-Dylight 594 conjugate (5 µg/mL each).