Sections were cut at 3 μm from individual paraffin-embedded blocks and immunostained for mouse monoclonal anti-αSMA (M0851, Dako; 1:400 dilution), mouse polyclonal anti-collagen-1 (AB34710, Abcam; 1:250 dilution) and polyclonal rabbit fibronectin (A0245, Dako; 1:1000 dilution) for 90 min at room temperature. Species-appropriate isotype-matched immunoglobulin G (X0931 clone DAK-GO1; Dako) was incorporated. Bound antibodies were elaborated using peroxidase-labelled Envision (K4001; Dako) and diaminobenzidine (K3468; Dako). In addition, we incorporated an overlapping group of smoker and COPD tissues from a previous study [15 (link)] in which SAs had been stained in the same way as described earlier for S100A4 and vimentin, both EMT activity markers in epithelium, where they are co-expressed with epithelial proteins.
Protocol detail
Immunostaining of Lung Tissue Sections
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Antibodies
Clone
Collagen 1
Copd
Dilution
Epithelium
Fibronectin
Immunoglobulin g
Isotype
Mouse
Mouse αsma
Paraffin
Peroxidase
Proteins
Rabbit
Tissues
Vimentin
Corresponding Organization :
Other organizations : University of Tasmania, University of British Columbia, St. Paul's Hospital, Deakin University, Royal Hobart Hospital, Newcastle University
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Variable analysis
independent variables
- Mouse monoclonal anti-αSMA (M0851, Dako; 1:400 dilution)
- Mouse polyclonal anti-collagen-1 (AB34710, Abcam; 1:250 dilution)
- Polyclonal rabbit fibronectin (A0245, Dako; 1:1000 dilution)
- Immunostaining of paraffin-embedded tissue sections
- Species-appropriate isotype-matched immunoglobulin G (X0931 clone DAK-GO1; Dako)
- Sections were cut at 3 μm from individual paraffin-embedded blocks
- Immunostaining was performed for 90 min at room temperature
- Positive control: Overlapping group of smoker and COPD tissues from a previous study [15 (link)] in which SAs had been stained for S100A4 and vimentin, both EMT activity markers in epithelium, where they are co-expressed with epithelial proteins
- Negative control: Species-appropriate isotype-matched immunoglobulin G (X0931 clone DAK-GO1; Dako)
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