X0931 clone dak go1

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.

Serial sections of resected tissue were immunostained for EMT mesenchymal biomarkers (S100A4, Vimentin), for the epithelial activation marker (epidermal growth factor receptor [EGFR]) and for vascularity using anti-Type-IV collagen for vessels in the Rbm. Sections of 3-µm thickness were mounted and stained with hematoxylin and eosin for morphological assessment and suitability for immunostaining. At room temperature, optimal sections were stained with the following antibodies: polyclonal anti-S100A4 (Dako, catalog no A5114, at 1:2,000 for 90 minutes), antivimentin monoclonal antibody (Dako, catalog no M7020, at 1:1,000 for 60 minutes), and Type-IV collagen monoclonal antibody (Dako, catalog no M0785, at 1:100 for 90 minutes) and monoclonal anti-EGFR (Dako, catalog no M3563, at 1:1,000 for 90 minutes). In each run, a section stained with immunoglobulin G1-negative control (X0931 clone DAKGO1; Dako Cytomation) was included to ensure absence of false-positive staining. Bound antibodies were elaborated by using horseradish peroxidase-conjugated DAKO Envision plus reagent (catalog no K4001, anti-mouse or K4003 anti-rabbit) and diaminobenzidine for a brown color resolution (catalog no K3468; Dako Cytomation). We have extensively used and published these methods.5 (link),6 (link),19 (link),20 (link)

Tissue was processed as previously described.3 (link),4 (link) Following removal of paraffin, followed by hydration, sections were stained with these antibodies: monoclonal-anti-MMP-9 (R&D Systems, Inc., Minneapolis, MN, USA; MAB911; 1:50 for 2 hours), monoclonal-anti-EGFR (Chemicon S/A, São Paulo, Brazil; CBL417; 1:40 for 60 minutes), and anti-S100A4 polyclonal antibody (Dako, Glostrup, Denmark; A5114; 1:2,500 for 90 minutes). In each case, a non-immune immunoglobulin (Ig)G1 negative control (Dako; X0931 clone DAK-GO1) was performed to eliminate false positive staining and a positive tissue control (surgically resected lung tissue) was also used. Bound antibodies were elaborated using peroxidase-labeled EnVision™ + (Dako; K4001) and liquid diaminobenzidine (DAB) + (Dako; K3468).