Ab6311

One drop of each sample of soluble protein was placed in a nitrocellulose membrane. After drying, membranes were incubated for 1 h with 5% BSA, with agitation, at room temperature. Subsequently, membranes were incubated with primary antibody:mouse antiCollagen type I 1:1000 (#ab90395, abcam, Cambridge, UK); rabbit antiCollagen type IV, 1:500 (#ab6311, abcam, Cambridge, UK); rabbit antifibronectin 1:500 (#ab45688, abcam, Cambridge, UK); and mouse antilaminin, 1:500 (#L8271, Sigma-Aldrich, St. Louis, MO, USA). After overnight incubation, membranes were washed 3× for 5 min with Tris Buffered Saline (TBS) with Tween 20 and then the R.T.U. VECTASTAIN^® Universal ABC Elite^® Kit (#PK-7200, Vector Laboratories, Burlingame, CA, USA) was used as a secondary antibody, in accordance with manufacturer’s instructions. Finally, incubation was revealed using a Peroxidase Substrate Kit (DAB) (#SK-4100, Vector Laboratories, Burlingame, CA, USA). Extraction buffer without samples was used as a negative control. Collagen type I (#sc-136157, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Collagen type IV (#C5533, Sigma-Aldrich), fibronectin (#FC010, Sigma-Aldrich, St. Louis, MO, USA) and laminin (#L6274, Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls. At least three independent samples were used in each condition.

Immunolocalization of different proteins, such as collagen type I, collagen type IV, fibronectin and laminin, was performed in paraffin-embedded samples sectioned at 5 μm, as previously described for collagen type II [30 (link)]. Briefly, samples were incubated with mouse anticollagen type I 1:100 (#ab90395, abcam, Cambridge, UK); rabbit anticollagen type IV, 1:50 (#ab6311, abcam, Cambridge, UK); rabbit antifibronectin 1:300 (#ab45688, abcam, Cambridge, UK); and mouse antilaminin, 1:300 (#L8271, Sigma-Aldrich, St. Louis, MO, USA), overnight at 4 °C in a humidified atmosphere. As a secondary antibody, the R.T.U. VECTASTAIN^® Universal ABC Elite^® Kit (#PK-7200, Vector Laboratories, Burlingame, CA, USA) was used, in accordance with manufacturer’s instructions. Incubation was revealed using a Peroxidase Substrate Kit (DAB) (#SK-4100, Vector Laboratories, Burlingame, CA, USA). Samples were counterstained with hematoxylin and mounted in an aqueous mounting medium. Slides were observed in an optical microscope with a coupled camera (DM750, Leica, Wetzlar, DE, Germany). At least three independent samples were used in each condition.

Immunofluorescence was performed on paraffin-embedded (FFPE) renal biopsies during renal flare (N = 3 for each subgroup) following the protocol described by Mason et al. [40 (link)]. Staining was performed with 1:50 rabbit anti-SP1 (Abcam, ab124804), 1:100 mouse anti-COL1A1 (Abcam, ab6308), or 1:100 mouse anti-COL4A1 (Abcam, ab6311) overnight at 4 °C (more details in SI).