For labeling fibroblast-like cells, the polyclonal antibody antiS100a4 ab27957 (Abcam, Cambridge, UK) was used.
Transwell cultures were embedded after 10 and 21 days in Tissue-Tek (Sakura, Zoeterwoude, The Netherlands) and frozen at − 20 °C. Samples were cut into 10 µm serial-sections, mounted on superfrost glass slides (Menzel, Braunschweig, Germany), fixed in acetone, and dried at room temperature. After re-hydrating with 0.2% Tween (Merck, Darmstadt, Germany) in PBS sections were incubated for 1 h at RT with ABO-Serum (Biotest, Dreieich, Germany) mixed with horse serum (both 1:20 in PBS with 3% Bovine Serum Albumin (BSA; Sigma-Aldrich Co.) to block unspecific binding sites. Incubation with the first antibody (AB) anti-S100A4 (Abcam) (1:100 in AB dilution buffer (DCS Innovative, Hamburg, Germany)) for 1 h at RT was performed. After washing, incubation with the biotinylated goat anti-rabbit AB (Vector Laboratories, Burlingame, USA) (1:200 dilution with AB dilution solution) was performed for 30 min at RT. After washing, the sections were treated with avidin–biotin–peroxidase complex (ABC; Vector Laboratories) for 30 min at RT. Then, the sections were washed. Staining was developed using 3-Amino-9-Ethyl Carbazol (Sigma-Aldrich Co.) and counterstained with hemalaun. The sections were then embedded in Aquamount (Merck, Darmstadt, Germany).
Transwell cultures were embedded after 10 and 21 days in Tissue-Tek (Sakura, Zoeterwoude, The Netherlands) and frozen at − 20 °C. Samples were cut into 10 µm serial-sections, mounted on superfrost glass slides (Menzel, Braunschweig, Germany), fixed in acetone, and dried at room temperature. After re-hydrating with 0.2% Tween (Merck, Darmstadt, Germany) in PBS sections were incubated for 1 h at RT with ABO-Serum (Biotest, Dreieich, Germany) mixed with horse serum (both 1:20 in PBS with 3% Bovine Serum Albumin (BSA; Sigma-Aldrich Co.) to block unspecific binding sites. Incubation with the first antibody (AB) anti-S100A4 (Abcam) (1:100 in AB dilution buffer (DCS Innovative, Hamburg, Germany)) for 1 h at RT was performed. After washing, incubation with the biotinylated goat anti-rabbit AB (Vector Laboratories, Burlingame, USA) (1:200 dilution with AB dilution solution) was performed for 30 min at RT. After washing, the sections were treated with avidin–biotin–peroxidase complex (ABC; Vector Laboratories) for 30 min at RT. Then, the sections were washed. Staining was developed using 3-Amino-9-Ethyl Carbazol (Sigma-Aldrich Co.) and counterstained with hemalaun. The sections were then embedded in Aquamount (Merck, Darmstadt, Germany).