Anti type 1 collagen

Paraffin-embedded mouse kidney sections (3-µm thickness) were stained with PAS (catalog no. G1280; Solarbio, Beijing, China), Masson (HT15-1KT; Sigma–Aldrich, St Louis, MI, USA), and Sirius red (catalog no. G1472-2; Solarbio, Beijing, China). The antibodies for immunohistochemistry were: anti-α-SMA (catalog no ab32575, Abcam, Cambridge, United Kingdom), anti-fibronectin (catalog no. 610154; Transduction Laboratories, Lexington, KY, USA), anti-type I collagen (catalog no. AB765P; Millipore Corp., Billerica, MA, USA) and anti-CCR2 (catalog no. ab176390; Abcam, Cambridge, United Kingdom) and anti-galectin-3 (catalog no. 3027070; Millipore Corp., Billerica, MA, USA). After incubation with the primary antibodies at 4 °C overnight, the slides were stained with the secondary antibody for 1 h at room temperature. The sections were incubated with the ABC reagents for 1 h at room temperature before DAB staining (Vector Laboratories, Burlingame, CA, USA). Images were captured using a light microscope (Olympus, Tokyo, Japan).

Kidney samples were fixed in 10% neutral formalin and embedded in paraffin. 3 μm-thick sections were used for Periodic Acid–Schiff (PAS) and Masson staining. For immunohistochemical staining, paraffin embedded kidney sections were deparaffinized, hydrated, and antigen-retrieved. Endogenous peroxidase activity was quenched by 3% H2O2. Sections were then blocked with 10% normal donkey serum, followed by incubation with anti-type I collagen (cat: AB765P, Millipore, Billerica, MA) over night at 4 °C. After incubation of secondary antibody for 1 h, sections were incubated with ABC reagents for 1 h at room temperature before being subjected to substrate 3-amino-9-ethylcarbazole or 3,3′diaminobenzidine (Vector Laboratories, Burlingame, CA). Slides were viewed with a Nikon Eclipse 80imicroscope equipped with a digital camera (DS-Ri1, Nikon, Shanghai, China).

Cultured NRK-49F cells were lysed in 1 × sodium dodecyl sulfate sample buffer. The kidneys were lysed with radio immunoprecipitation assay (PIPA) solution containing 1% NP40, 0.1%sodium dodecyl sulfate, 100 mg/ml phenylmethanesulfonylfluoride, 1% protease inhibitor cocktail, and 1% phosphatase I and II inhibitor cocktail (Sigma, St Louis, MO) on ice. The supernatants were collected after centrifugation at 13,000 g at 4 °C for 30 min. Protein concentration was determined by bicinchoninic acid protein assay (BCA Kit; Pierce Thermo-Scientific, Rockford, IL) according to the manufacturer’s instructions. Equal amount of protein was loaded into 10 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes. The primary antibodies were as follows: anti-GAPDH (cat: FL-335, Santa Cruz Biotechnology, Dallas, TX), anti-p-Akt (Ser473) (cat: 3868, Cell Signaling Technology), anti-p-Akt (Thr308) (cat: 2965, Cell Signaling Technology), anti-p-Smad3(Ser423/425) (cat: ab52903, Abcam), anti-FN (cat: F3648, Sigma-Aldrich), anti-α-SMA (cat: A5228, Sigma-Aldrich), and anti-type I collagen (cat: AB765P, Millipore). Quantification was performed by measuring the intensity of the signals with the aid of National Institutes of Health Image J software package.

A series of 5-μm thick cryosections of cPDLSC sheets were prepared and incubated at room temperature for 60 min in 50 mM Tris-buffered saline with 0.4% Triton X-100 (TBS-T; pH 7.2) containing 5% bovine serum albumin (BSA). For the detection of fibronectin, the cells were stained overnight with 1:500 anti-fibronectin (Sigma-Aldrich) diluted in TBS-T containing 1% BSA. After washing to remove the unbound primary antibodies, the cells were then incubated at room temperature for 1 h with 1:200 fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin M (Chemicon, USA) diluted in TBS-T containing 1% BSA. For the detection of integrin, 1:500 anti-integrin (Sigma-Aldrich) and 1:200 FITC-labeled goat anti-mouse immunoglobulin M (Chemicon) were used as the primary and secondary antibodies, respectively. For the detection of collagen type I, 1:1000 anti-collagen type I (Sigma-Aldrich) and 1:200 phycoerythrin-labeled goat anti-mouse immunoglobulin G (Chemicon) were used as the primary and secondary antibodies, respectively. The fluorescently labeled cell sheets were viewed under a confocal laser scanning microscope (Carl Zeiss LSM700, Germany).

Fresh and glutaraldehyde fixed pericardial samples were embedded in Optimal Cutting Temperature material and cryosectioned (8 μm). Sections were air dried, fixed with acetone for 10 min and stored at −80 °C until used. Fresh and glutaraldehyde fixed samples were also fixed in formalin and paraffin embed for Masson’s Trichrome histology staining. Tissue sections were stained to detect the Gal antigen and collagens. Fresh and glutaraldehyde fixed tissues were stained with biotin-conjugated GSIB-4 (3 μg/ml) and murine monoclonal antibodies (2 μg/ml) to Collagen I (Anti-Collagen Type I, Sigma-Aldrich), III (Anti-Collagen Type III, Abcam, Cambridge, UK), and V (Anti-Collagen Type V, Abcam). Lectin and antibody solutions were produced in dPBS with 1% BSA and primary incubations were at 4 °C for 12 h. GSIB-4 binding was detected with HRP-conjugated streptavidin (Sigma-Aldrich) and mouse anti-collagen antibody binding was detected with biotin conjugated goat anti-mouse IgG (Sigma-Aldrich) and HRP-conjugated streptavidin. All sections were incubated with 3,3′-Diaminobenzidine (Sigma-Aldrich) and counter stained with haematoxylin.