Bovine serum albumin (bsa)

Respiratory syncytial virus (RSV) strain and human epidermis larynx carcinoma cell lines (HEp-2 cells) were cultivated and obtained in our own laboratory. Streptavidin (SA) was purchased from Beijing Boisynthesis Biotechnology Co. Ltd. (Beijing, China). Bovine serum albumin (BSA) was purchased from Beijing Dingguo Changsheng Biotechnology Co. Ltd. (Beijing, China). Biotin was purchased from Thermo (U.S.A). Streptavidin−quantum dot (QD) conjugates were purchased from Jiayuan Quantum Dot Co. Ltd. (Wuhan, China). Other commercial reagents such as HAuCl₄ and trisodium citrate were analytical reagent grade without further purification. All the above reagents were prepared with Milli-Q purified water (18.2 MΩ).

Total cell lysates were prepared using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) and the supernatant was collected via centrifugation at 4°C and 4,024 × g for 10 min. A bicinchoninic acid assay was used to determine the protein concentration. Aliquots (30–40 µl) of cell lysates were heated at 100°C for 5 min, and 10 µg of protein was loaded into each well of a 10% SDS-PAGE gel for electrophoresis. The proteins on the electrophoresis gel were then transferred to an Immobilon-P membrane that was incubated in blocking solution [5% bovine serum albumin (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) in TBS-Tween 20] for 1–3 h at 25°C followed by overnight incubation at 4°C with mouse monoclonal anti-human CD105, CD44 and CD106 antibodies at dilutions of 1:1,000. Subsequent to washing 3 times in TBS with 0.1% Tween 20, the membrane was incubated for 1–2 h at 25°C with a fluorophore-conjugated secondary antibody (cat. no. 610-132-121; Rockland Immunochemicals, Inc., Limerick, PA, USA) at a dilution of 1:5,000. The membrane was washed and analysed using an Odyssey two-colour infrared imaging system (LICOR Odyssey, LI-COR Biosciences, Lincoln, NE, USA). The signal intensity of protein bands was calculated using Image J software (v1.8.0; National Institutes of Health, Bethesda, MD, USA).

PASMCs were fixed in medium containing 4% paraformaldehyde for 30 min. The cells were permeabilized using 0.2% Triton X-100 for 2 min, and were blocked with 5% bovine serum albumin (Dingguo Changsheng Biotechnology, Co., Ltd.) for 30 min. The cells were then incubated with α-SMA antibody (Beyotime Institute of Biotechnology) at 4°C overnight, were washed with PBS, and were incubated with streptavidin biotin complex-fluorescein isothiocyanate (Wuhan Boster Biological Technology, Ltd.) for 30 min at 37°C. After counterstaining with 4′,6-diamidino-2-phenylindole (DAPI), immunofluorescence was observed and images were captured under a fluorescence microscope (IX73-A22FL/PH).

The kidney paraffin sections, prepared as described in the previous section, were dewaxed at 60˚C for 30 min, and washed with boiled 0.01 mol/l PBS (pH 7.4, 0.0203 mol/l Na₂HPO₄, 0.00167 mol/l NaH₂PO₄, 0.1367 mol/l NaCl) three times (3 min each), blocked in 5% bovine serum albumin (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) at room temperature for 1 h. Afterwards, the samples were incubated with rabbit anti-tTG antibody for 24 h at 4˚C (1:300 dilution), the bound antibodies were subsequently detected with HRP-anti-rabbit IgG for 20 min at room temperature (1:1,000 dilution) and DAB, followed by counterstaining with Mayer's hematoxylin for 2 min at room temperature, and negative controls were incubated with PBS. Finally, images were captured under a light microscope (Nikon TE-2000U; Nikon Corporation), and analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Inc.) analysis software.