Microscopy

Myocardial tissues were routinely paraffin-embedded and sectioned at 4 μm. The sections were first dewaxed and rehydrated. After blockage of endogenous peroxidase with 3% hydrogen peroxide solution, the sections were blocked with 100 μL of 5% BSA at 37 °C for 0.5 h. The sections were incubated overnight in 50 μL antibody against α-SMA (1:500, GTX100034, GeneTex) at 4 °C and with biotin-labeled secondary antibody goat anti-rabbit working solution (1:2000, ab205718, Abcam) at 37 °C for 0.5 h. Then, the sections were incubated with streptavidin-peroxidase solution (Solarbio, Beijing, China) for 0.5 h at 37 °C. Finally, the sections were treated with diaminobenzidine, counter-stained with hematoxylin for 30 s, dehydrated, fixed, observed and counted under a microscopy (Olympus Optical Co., Ltd., Tokyo, Japan).

The paraffin blocks were cut into sections of 4 μm, mounted on polylysine-coated microslides, dewaxed, and rehydrated. Then, tissue sections were incubated in 3 % hydrogen peroxide at room temperature. For antigen retrieval, tissue slides were immersed in citric acid and boiled in a microwave oven. The sections were washed in distilled water and phosphate buffered saline (PBS, Gibco), and then subjected to bovine serum to block unspecific binding agents. This step was followed by overnight exposure (4 °C) to the mouse anti-human Gal-3 antibody (mab1154, R&D Systems Inc., Minneapolis, MN, USA) in a humidified chamber. After being rinsed in PBS, the sections were incubated with the bridging rabbit anti-mouse immunoglobulins conjugated with horseradish peroxidase (HRP)-labelled dextran polymer for 1 h. After washing in PBS, diaminobenizidine tetrahydrochloride (DAB) solution was applied, followed by running tap water as well as nuclear counterstaining with haematoxylin (Sigma, USA). The stained slides were viewed under microscopy (OLYMPUS OPTICAL CO., Ltd.) under 400× magnification. Positive cells were characterized by the brown staining of Gal-3 antibody. The intensity and distribution of the staining reaction were evaluated by two blinded, independent observers.

The lung tissue sections were incubated with 1 : 200 diluted mouse polyclonal antibodies against phospho-NF-κBp65 and NOX1 overnight at 4°C in a humidified chamber. Then, these immune complexes were incubated with the appropriate secondary antibody for 20 min at room temperature before being rinsed 3 times with PBS. Immunoreactivity was represented by brown staining using DAB (Beijing Biosynthesis Biotechnology Co., Ltd.). The sections were subsequently washed with distilled water, counterstained with H&E, and photographed via microscopy (Olympus Optical Co., Tokyo, Japan). The IHC staining results were evaluated by independent pathologists who were blinded to the study. Immunohistochemical staining intensity was scored using a scale ranging from 0 to 3 (negative = 0, weak = 1, moderate strong = 2, or strong = 3). Staining extent was assessed based on the percentages of positive cells as follows: 0 (negative), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). The final staining score was calculated as the mean of the sums of the scores in five fields in every section, and all the sections were separated into low expression groups (final score = 1–5) and high expression groups (final score = 6–12).

RNeasy FFPE Kit (cat. no. 73504) was purchased from QIAGEN Ltd (Hilden, Germany). Trizol was obtained from Invitrogen CO., Ltd. (Carlsbad, California, USA). Reverse transcription Kit (#K1622) and real-time fluorescent quantitative PCR Mix (#K0221) were purchased from Thermo Fisher Scientific Inc (Vilnius, Lithuania). The specific reverse transcriptase and PCR primers of U6 and miR-143 were synthesized by Taihe Biotechnology Co., Ltd. (Beijing, China). The bcl-2 antibody (sc-7382) was purchased from Santa Cruz Biotechnology Inc (California, USA). The universal secondary antibody (PV-6000), goat serum and diaminobenzidine (DAB) reagent were all purchased from Beijing Zhong Shan-Golden Bridge Biological Technology CO., Ltd. (Beijing, China). Cell lysis buffer and the HPV genotype detection kit were products from Chaozhou Kaipu Biochemistry Co. Ltd. (Chaozhou, China). ABI7500 real-time PCR system was purchased from Applied Biosystems, Inc. (New York, USA), and the OLYMPUS microscopy was from Olympus optical Co., Ltd. (Tokyo, Japan).

Briefly, tissue samples were collected, mounted onto slides from paraffin blocks (5-μm sections), and then deparaffinized in xylene, followed by hydration in a methanol gradient (100%, 95%, 70%, and 50%). Slide samples were treated and 10 mM citrate buffer (pH 6.0) containing 3% H₂O₂ for antigen retrieval and then incubated with 5% BSA for 30 min. The slides were incubated with the primary antibody at 4°C overnight and then incubated with the biotinylated secondary antibody for 30 min. After that, an avidin-biotin complex kit (Dako/Agilent Technologies, Santa Clara, CA, USA) was used for an additional 30 min, and 3,3′-diaminobenzidine tetrahydrochloride hydrate (DAB) containing 5% H₂O₂ was used as a chromogen. Hematoxylin and eosin (H&E) staining was performed and visualized by microscopy (Olympus, Tokyo, Japan). The antibodies used are as follows: mouse anti-neurofilament L (clone DA2) (CST, USA), anti-4-HNE (Abcam), anti-CD11b (Abcam), anti-IBA-1 (CST), and anti-GFAP (CST). The intensity of immunohistochemistry (IHC) or immunofluorescence (IF) staining was analyzed using ImageJ software.

Liver tissues were embedded in TissueTek optimal cutting temperature (OCTTM) compound (Sakura Finetek) and frozen at −80°C for storage. Frozen sections (4 μm thick) were prepared using a cryostat (CM3050 S, Leica) and processed for Sirius Red staining. Cryosections were fixed with an isotonic PBS and 4% paraformaldehyde solution for 1 h. To block non-specific background staining, the samples were incubated in a solution containing 1% BSA for 30 min. After washing with PBS, the slides were incubated with the primary antibodies. Anti-glial fibrillary acidic protein (GFAP) (ab10062, abcam), anti-HDAC4 (#5392, cell signaling, MA), and anti-a-SMA (ab5694, abcam, UK) primary antibodies were used. Fluor 488-conjugated (green) and Alexa Fluor 595 (red)-conjugated secondary antibodies (Molecular Probes) were used. Samples were co-stained with 4ʹ,6-diamidino-2-phenylindole (DAPI; Molecular Probes) to facilitate visualization of the nuclei. The stained cells were mounted with a fluorescent mounting medium (Dako Cytomation) and visualized by microscopy (Olympus). The exposure gains and rates were consistent between samples. Fluorescent intensities were quantified on independent color channels by using Image J analysis.