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).
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).
Free full text: Click here
