Ab11575

Kidneys of sacrificed mice were kept in 4% paraformaldehyde at 40 C overnight and immersion-fixed using HistoCore PEARL (Leica, Wetzlar, Germany). Paraffin-embedded kidneys were sectioned at 3 μm on a Reichert-Jung 2040 Autocut Microtome (Leica Microsystems, Wetzlar, Germany). For staining of laminin 111 and fibronectin, the slides were de-paraffinized and rehydrated. Sections were incubated overnight at 4 °C with the primary antibodies rabbit anti-laminin 111 (1:25, Ab11575, Abcam, Cambridge, UK), and goat anti-fibronectin (1:50, sc6952, Santa Cruz Tech, Dallas, TX, USA). As a negative control, slides were incubated with blocking solution (5% donkey serum in Tris-buffered saline). After washing the samples were stained with the secondary antibody. The Alexa 488-labled antibody goat anti-rabbit (A11008, Lifetech, Waltham, MA, USA) was used to prepare the laminin 111 staining and the donkey anti-goat (A11055, Lifetech, Waltham, MA, USA) was added to the slides for fibronectin staining. Both antibodies incubated for one hour at room temperature in the dark. After washing all slides were mounted in fluorescence mounting medium (S303, Dako, Waldbronn, Germany).

Five µm thick tissue sections from dehydrated and paraffin-embedded MMPs treated and untreated dUTDs were mounted on slides and processed for Russel-Movat pentachrome staining using a standard staining kit (KSC-MPS-2, Nordic BioSite, Täby, Sweden). Sections from each scaffold type were also processed for immunohistochemistry. An antigen retrieval step was first conducted using citric acid (pH = 6.0) in a pressure cooker. The sensitive Mach 3 and vulcan fast red kits (Biocare Medical, Pacheco, CA, USA) were then used to visualize collagen I (#ab292), collagen IV (#ab6586), laminin (#ab11575), elastin (#ab23748), and fibronectin (#ab6328) using antibodies from Abcam (Cambridge, UK) at a concentration of 1:100.

Tissue samples were fixed overnight in 2% paraformaldehyde at 4°C and subsequently processed for paraffin embedding. Bone samples have been decalcified through 20% EDTA prior embedding. 7 μm thick sections were obtained through a Leica RM2155 microtome. The slides have been deparaffinized, rehydrated and subjected to the following staining: Hematoxilin and Eosine (Sigma Aldrich); Masson’s Trichrome (American Master Tech); Verhoeff’s elastic stain (American Master Tech). For immunofluorescent analysis, we performed antigen retrieval on deparaffinized sections using a citrate-based antigen unmasking solution (Vector Laboratories; H-3300). The sections were blocked with 1%BSA and 1% Donkey Serum (Sigma Aldrich) and incubated overnight at 4 C with the following primary antibodies: mouse anti-myosin (DSHB; MF20); mouse anti Sarcomeric alpha-actinin (Sigma Aldrich; SAA A7811); rabbit anti Laminin (Abcam; ab11575); rabbit anti Collagen IV (Abcam; ab6586); mouse anti Neurofilament H (Sigma Aldrich, NF200 ab5539); rabbit anti S100 calcium binding protein B (Abcam; ab52642). After primary antibody binding, the sections were washed and incubated for one hour at room temperature with the appropriate donkey-raised secondary antibodies, conjugated with a 488, 546 or 647 fluorochromes (Life Technologies; AlexaFluor series).

Whole-mount primary antibody labeling was performed as described above. After overnight incubation at 4°C with a rabbit pAb anti-laminin antibody (ab11575, Abcam), organoids were washed with PBS-Triton and incubated overnight at 4°C with a goat anti-rabbit IgG labeled with 10 nm gold (ab39601, Abcam) diluted 1:400. Samples were then fixed with 4% paraformaldehyde and 2.5% (wt/vol) glutaraldehyde (Agar Scientific, UK) in 0.1 M HEPES (H0887, Sigma-Aldrich) pH 7.2, and postfixed with 1% osmium tetroxide (R1024, Agar Scientific) and 1.5% potassium ferrocyanide (214022, The British Drug House, Laboratory Chemicals Division) in 0.1 M cacodylate buffer (R1102, Agar Scientific) pH 7.2 for 1 hr, then with 1% uranyl acetate (R1100A, Agar Scientific) in water for overnight. Samples were dehydrated, embedded with low-viscosity medium-grade resin (T262, TAAB Laboratories Equipment Ltd) and polymerized for 24 hr at 60°C. For transmission electron microscopy, sections were cut with a Reichert Ultracut ultramicrotome and observed with a FEI Tecnai 12 Biotwin microscope at 80 kV accelerating voltage equipped with a Gatan Orius SC1000 CCD camera.

ASCs were stained after 1 and 14 days of culture. The cells were washed three times with PBS and fixed with 4% formaldehyde for 10 min at room temperature. The cells were then permeabilized with 0.3% Triton X-100 for 2 min. The cells were blocked in 1% BSA for 30 min, then incubated with anti-fibronectin (ab2413; Abcam) and anti-laminin (ab11575; Abcam) primary antibodies overnight at 4°C. Then, the cells were incubated with secondary antibodies and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). F-Actin was stained with phalloidin (Sigma-Aldrich) for 45 min.

Cultured microglia were fixed with 4% paraformaldehyde at room temperature for 15 minutes, blocked with 0.5% bovine serum albumin/0.1% Triton X-100 in PBS and stained with anti-laminin (Ab11575, Abcam), anti-CD11b (MCA74G, Serotec), anti-IBA1 (019–19741, Wako), anti-Adam9 (AF949, R&D System), and anti-Ctss (sc-271619, Santa Cruz) antibodies overnight at 4 °C. After being washed with PBS, the cultures were incubated with secondary antibodies conjugated to Alexa Fluor (Invitrogen) at room temperature for one hour. The nuclei were subsequently counterstained with DAPI.