Immunofluorescence stainings were performed on 6 μm frozen sections, and on cells cultured on Falcon™ Culture Slides (BD Biosciences, Erembodegem, Belgium) using methods previously described [
20], with the following primary antibodies and dilutions: mouse anti-human integrin α
1 (MAB1973, 1:40); mouse anti-human integrin α
2 (MAB1950, 1:40); mouse anti-human integrin β
1 (MAB1959, 1:500), all from Millipore (Billerica, MA, USA); and rabbit anti-human type IV collagen (MP Biomedicals, LLC, Solon, OH, USA; cat. no. 10760, 1:100); goat anti-human type XVIII collagen (R&D Systems, Minneapolis, MN, USA; AF570, 1:75), goat anti-human perlecan (R&D Systems, Minneapolis, MN, USA; AF2364, 1:75), rat anti-human laminin-γ1 (Millipore, Billerica, MA, USA, Mab1914P, 1:75), rabbit anti-human nidogen (Millipore, Billerica, MA, USA, cat. no. 481978, 1:75), mouse anti-human cytokeratin 18 (DakoCytomation, Glostrup, Denmark, DC10 1:100) and sheep anti-CD31 (R&D Systems, Inc., Minneapolis, MN, USA; AF806, 1:50). Cultured cells were stained with the integrin antibodies mentioned above together with the goat anti-type IV collagen antibody (Chemicon, Billerica, MA, USA; AB769, 1:50). Double staining on tissue for type I and type IV collagen was performed with a rabbit anti-human type I collagen antibody (Cedarlane Laboratories, Burlington, NC, USA; CL50111AP, 1:200) and a mouse anti-α1(IV)NC1 (Wieslab, Malmö, Sweden; MAB1, 1:75) [
21]. Secondary antibodies used were: donkey anti-rabbit FITC, donkey anti-mouse TRITC and FITC, donkey anti-goat TRITC (all Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; with dilutions 1:100). Sections were mounted with medium containing DAPI (Vectashield, Vector Laboratories, Inc., Burlingame, CA, USA). Negative control sections were incubated with secondary antibodies only. Hematoxylin and eosin (H&E) stainings were performed according to standard protocols.