Horseradish peroxidase conjugated goat anti mouse antibody

Liver tissues were fixed in 4% paraformaldehyde for 12 h, embedded in paraffin and cut into 5-μm sections. Sections were stained with hematoxylin and eosin (HE), picric acid-sirius red (PSR), and Masson, for histological structure analysis and fibrosis area analysis. Five randomly selected fields of view, from PSR- and Masson-stained sections of each sample (n = 3/group), were captured by a light microscope (Olympus, Tokyo, Japan).The fibrosis area was measured using Image-Pro Plus software (Media Cybernetics). The percentage fibrosis area was calculated by comparing the collagen stained area to the total area of the fields examined.
Immunohistochemical (IHC) staining was carried out as described previously using commercially available antibodies against collagen I, α-smooth muscle actin (α-SMA), Ki-67 (all from Abcam), and CD31 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). This was followed by horseradish peroxidase-conjugated goat anti-mouse antibody (Dako, Denmark) and colorized with diaminobenzidine tetrahydrochloride (DAB, Dako) [8] (link). For blood vessel density analysis, five randomly selected fields, from anti-CD31 staining of each sample (n = 3/group), were captured by a light microscope (Olympus).The number of blood vessels was calculated by Image-Pro Plus software (Media Cybernetics).

Rituximab concentration in samples collected just before and just after each infusion was measured using an enzyme-linked immunosorbent assay. 96-well ELISA MaxiSorp plates (ThermoFisher Scientific) were coated with 1 µg/ml of anti-Rituximab (anti-idiotype) antibody RB01 (R&D Systems) and blocked with washing buffer (50 mM Tris-HCl, 0.15 M NaCl, 0.1% Tween, pH 7.5) supplemented with 3% fish skin gelatin (Sigma-Aldrich). Patients’ serum was diluted to the final concentration of 0,125% in PBS with 0.02% Tween-20 and 0.02M EDTA. Rituximab (Roche) serially diluted in NHS was used for the preparation of the calibration curve. The horseradish peroxidase-conjugated goat anti-mouse antibody (Dako, P0447) was used for detection. The assay was developed using 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma-Aldrich), and absorbance readout at 450 nm was measured using a Synergy H1 microplate reader (Biotek).

Dulbecco's modified Eagle's medium (DMEM), penicillin, and streptomycin were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Fetal bovine serum (FBS) was obtained from GE Healthcare Life Sciences (Logan, UT, USA). UVB light (Philips 311 nm, TL 20W/01) was from Philips Lighting Holding B.V. (Eindhoven, The Netherlands). Cell Counting Kit-8 (CCK-8) was from Beyotime Institute of Biotechnology. RNase A, propidium iodide, FITC-phalloidin, and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH₂-DA) were from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Senescence-associated β-galactosidase (SA-β-gal) staining kit (#9860) was from Cell Signaling Technology Inc. (Danvers, MA, USA); terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP-biotin nick end labeling (TUNEL) staining kit was from Roche Molecular Biochemicals (Mannheim, Germany). Anti-CD31 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated goat anti-mouse antibody was from Dako (Glostrup, Denmark). Anti-GPX-1 (#ab108427), anti-catalase (#ab16731), anti-SOD-2 (#ab13533), anti-SOD-1 (#ab13498), anti-COL-1 (#ab6308), and β-actin (#ab6276) were from Abcam (Cambridge, UK); horseradish peroxidase-conjugated goat anti-mouse secondary antibody (#115-035-062) was from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA).

A 10-fold dilution of the collected apical washings and basolateral chamber media were distributed onto Vero cells (flaviviruses) or HEp-2 cells (RSV) and maintained for 72 h at 37°C and 5% CO₂. For flavivirus detection, the cells were washed with PBS, fixed with 4% PFA, and incubated with anti-E protein Flavivirus group antibody 4G2 in PBS supplemented with 0.3% saponin (Sigma-Aldrich), followed by 30 min incubation with horseradish peroxidase-conjugated goat anti-mouse antibody (Dako) and staining with 3-amino-9-ethylcarbazole substrate (Sigma-Aldrich). For RSV detection, after 72 h, the cells were washed with PBS, fixed with methanol supplemented with 2% H₂O₂, and incubated with a biotinylated anti-RSV antibody (Biorad) in PBS containing 1% albumin from bovine serum (Sigma) for 1 h, followed by 30 min incubation with ExtrAvidin Peroxidase (Sigma) and staining with 3,3′-diaminobenzidine substrate (Sigma). Titers were determined by counting plaques and expressed as PFU/ml.

Cell supernatants were collected 24, and 48 h p.i. and stored at −70 °C. Different 10-fold dilutions of cell supernatants were spread onto Vero cells and maintained for 72 h at 37 °C, 5% CO₂. Next, the cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) and incubated with anti-flavivirus group antibody 4G2 (ATCC, HB-112™) in PBS supplemented with 0.3% saponin (Sigma-Aldrich), followed by horseradish peroxidase-conjugated goat anti-mouse antibody (Dako) incubation for 30 min and staining with 3-amino-9-ethylcarbazole substrate (Sigma-Aldrich). Titers expressed as TCID₅₀/ml were determined by using the Reed and Muench method⁵⁴ .

The influenza A viruses (IAV) used were A/swine/Belzig/2/01 (H1N1) (Belzig/01) and A/swine/Bakum/R757/2010 (H1N2) (R757/10), both kindly provided by the Friedrich-Loeffler-Institute, Greifswald-Insel Riems, Germany. IAV were propagated in 10-day-old embryonated specific-pathogen-free (SPF) chicken eggs. Following incubation of the infected embryos for 2 days at 37 °C, the allantoic fluid was collected and IAV titrated on confluent MDCK cells in the presence of 1 μg/ml of acetylated trypsin (Sigma-Aldrich). The cells received new medium at 24 h post infection (p.i.) and at 48h p.i. were fixed with 4% paraformaldehyde (Polysciences, Warrington, PA, USA) followed by permeabilization with PBS 0.3% saponin (Sigma-Aldrich) in presence of NP antibody (HB-65, ATCC) for 15 min. Then, horseradish peroxidase-conjugated goat anti-mouse antibody (Dako, Baar, Switzerland) was added, and a final color reaction using 3-amino-9-ethylcarbazole (AEC, Sigma-Aldrich) and H₂O₂ as substrate. Titers were calculated using the Reed and Muench formula^{38 (link)}.