Anti collagen type 4

Extracellular matrix expression of the renal cortex in cryostat sections was identified by fluorescent staining using a specific anti-fibronectin (Abcam), anti-collagen type IV (Abcam), anti-alpha-smooth muscle actin (Abcam), anti-Gr1 (BD Biosciences) antibody, following permeabilization with 0.05% saponin buffer using a Vectastain Elite ABC kit (Vector Lab, Inc., Burlingame, CA, USA) as immunoperoxidase. The slides were developed using AEC Chromogen/Substrate and counterstained with hematoxylin. Isotype and species-matched irrelevant antibodies served as controls.

Paraffin-embedded tissues were sectioned at 5 μm thickness. The intact and decellularized sections were stained with hematoxylin and eosin (H&E) and Hoechst (10 ng/mL in H₂O) to confirm effective cell removal. Periodic acid Schiff (PAS), Alcian blue, Masson's trichrome, and Aldehyde fuchsin staining were performed to evaluate ECM components preservation including neutral carbohydrate, acidic glycosaminoglycans, collagen, and elastic fibers.
To evaluate the retention of collagen type I and IV, laminin, and fibronectin, we prepared unfixed frozen sections that were immunostained with biotinylated anti-collagen type I (1:250), anti-collagen type IV (1:500), anti-fibronectin (1:250), and anti-laminin (1:100) antibodies overnight at 4 °C (All from Abcam PLC, Cambridge, MA, USA). The color development was done by treating the samples with 200–400 μL streptavidin-HRP (1:10,000; Abcam, USA; ab7403) followed by incubating in 3,3′-Diaminobenzidine tetrahydrochloride (Sigma Aldrich, D5905). Finally, the samples were counterstained with hematoxylin.

Citrus junos seed residue, a byproduct of the post-yuzu-juice-preparation process, was obtained from the Lotte R&D Center (Seoul, Republic of Korea). The following monoclonal antibodies were used: anti-tyrosinase-related protein-2 (TRP-2) (Abcam, Cambridge, UK), anti-collagen type IV (Abcam), and anti-filaggrin (Santa Cruz Biotechnology, Dallas, TX, USA). Anti-collagen type I antibody and all cell culture solutions were purchased from Invitrogen (Carlsbad, CA, USA).
For Western blotting, antibodies that act against tyrosinase, TRP-1, TRP-2, and MITF were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), whereas antibodies that act against α-MSH, melanin, and β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Permeabilization was performed with 1% Triton X-100-CB (Sigma-Aldrich) solution for 10 min at RT. Unspecific antibody binding was blocked with blocking buffer (0.1% BSA, 0.2% Triton X-100, 0.05% Tween 20, in CB; Sigma- Aldrich) containing 5% skim milk powder (Sigma-Aldrich), and 1% Goat Anti-Mouse IgG, F (ab')₂ fragment specific (20 μg/ml, Jackson ImmunoResearch, United Kindom) for 2 h at RT. Primary antibodies anti-type IV collagen (Abcam), anti-cleaved caspase-3 (Cell Signaling Technology) or anti-GM 130 (BD Biosciences) were diluted 1:200 in 1% blocking buffer in CB and incubated overnight at 4°C. Secondary antibodies coupled with fluorescent dyes were equally diluted and applied to samples for 45 min at RT in darkness. Before storing the samples in darkness at 4°C they were treated with Nuc Blue Fixed Cell Stain (Invitrogen) or DRAQ5™ (Cell Signaling) for 10 min and washed thrice with cold CB.

In paraffin-embedded kidney sections (3 µm), antigens were retrieved by the PTlink link system. After the slides were blocked with 10% BSA and 10% FBS for 1 h, they were incubated with anti-SOX9 (1/200; Millipore) or anti-type IV collagen (1:1000; Abcam) for 1 h, followed by an AlexaFluor^® 488 conjugated rabbit anti-mouse secondary antibody (1/200; Invitrogen); AlexaFluor^® 633 conjugated rabbit anti-mouse secondary antibody (1/200; Invitrogen) or AlexaFluor^® 568 conjugated rabbit anti-mouse secondary antibody (1/200; Invitrogen) for 1 h. The absence of a primary antibody was used as a negative control. Samples were mounted in prolong gold (Thermo Fisher Scientific; Waltham, MA, USA) and examined using a Leica DM-IRB confocal microscope.