3 amino 9 ethylcarbazole aec

Virus isolations from ovine samples were performed by serial dilution of either plasma in complete CIM supplemented with 3,5 IU/ml heparin, or organ suspension in CIM, followed by incubation with BHK-21 cells. After 1.5 h incubation at RT, the inoculum was replaced by fresh medium and after 5 days of culturing the cells at 37°C cytopathic effects were scored.
Virus isolations from the supernatant of the human placenta explants were performed by a 10x serial dilution series of the supernatant in complete medium on Vero cells. At 24 hpi, infection was visualized with an immunoperoxidase monolayer assay (IPMA). Briefly, cells were fixed and permeabilized 24 hpi with 4% paraformaldehyde (10 min) and ice-cold methanol (5 min). After permeabilization the plates were incubated with RVFV specific monoclonal antibody 4-D4, which recognizes the Gn protein [17 (link)]. A polyclonal rabbit-α-mouse immunoglobin/HRP antibody (Dako, Denmark) was used as a secondary antibody and 3-Amino-9-ethylcarbazole (AEC; Sigma-Aldrich) was used as a substrate. The titre was expressed as TCID₅₀/ml according to the Spearman-Kärber algorithm [18 ,19 ].

Immunohistochemical staining was performed on neutral buffered formalin-fixed, deparaffinized arterial sections of warfarin-exposed and control rats sacrificed at week 10, as well as on calcified human arterial sections. The excised tissue sections were blocked with normal goat serum (20% in PBS) for 20 min and incubated overnight with polyclonal rabbit antisclerostin (1:500, ab-63097, Abcam, Cambridge, UK) or LRP4 (1:75, sc-98775, Santa Cruz, TX, USA). Biotinylated goat anti-rabbit (Vector Laboratories, Burlingame, CA, USA) was used as a secondary antibody. Avidin/biotinylated peroxidase complex (VECTASTAIN ABC kit, Vector Laboratories) was added as a signal amplifier and 3-amino-9-ethylcarbazole (AEC, Sigma-Aldrich, St. Louis, MO, USA) was used as a substrate. The sections were counterstained with hematoxylin. Sections in which the primary antibody was omitted were used as negative controls.

Formalin‐fixed, paraffin‐embedded tissue sections were rehydrated in series of alcohol from 100, 96, and 70% and lastly distilled water. Endogenous peroxidase activity was blocked with 1% hydrogen peroxide for 30 min at room temperature. Tissue slides were boiled in citrate buffer pH 6.0 for 10 min at 900 kW in a microwave for antigen retrieval and washed in PBS‐T for three times. Tissues were blocked with 3% BSA in PBS‐T overnight at 4°C. HAs were pre‐complexed with mouse anti‐strep‐tag‐ HRP antibodies (IBA) and goat anti‐mouse IgG HRP antibodies (Life Biosciences) in a molar ratio of 4:2:1 in PBS‐T with 3% BSA and incubated on ice for 15 min. After draining the slide, the pre‐complexed HA was applied onto tissues and incubated for 90 min at RT. Sections were then washed in PBS‐T, incubated with 3‐amino‐9‐ethyl‐carbazole (AEC; Sigma‐Aldrich) for 15 min, counterstained with hematoxylin, and mounted with Aquatex (Merck). Images were taken using a charge‐coupled device (CCD) camera and an Olympus BX41 microscope linked to CellB imaging software (Soft Imaging Solutions GmbH, Münster, Germany).

The viruses used are summarized in Table 1. All IAV were propagated in 10-day-old embryonated specific-pathogen-free (SPF) chicken eggs incubated for 2 days at 37°C. IAV were titrated on confluent MDCK cells in presence of 1 μg/ml of acetylated trypsin (Sigma-Aldrich). After 24 h, cells were fed by addition of new medium, and 48 h post-infection (p.i.), the cells were fixed with 4% paraformaldehyde (Polysciences, Warrington, PA, USA). The plates were stained with anti-NP antibody (HB-65, ATCC) in saponin (Sigma-Aldrich), followed by horseradish peroxidase-conjugated goat anti-mouse antibody (Dako, Baar, Switzerland), and a final color reaction with 3-amino-9-ethylcarbazole (AEC, Sigma-Aldrich). Titers were calculated using the Reed and Muench formula.

Murine IFN-γ ELISpot assays were purchased (ebioscience, Frankfurt, Germany) and performed according to manufacturer´s instructions using Multiscreen-IP ELISPOT PVDF 96-well plates (Millipore, Darmstadt, Germany). For this purpose, 5 × 10⁵ splenocytes isolated 4 d after booster immunization were co-cultured with 5 × 10⁴ JAWSII_green- or DC2.4_green-cells transgenic for H7 or N9, or the unmodified DC cell lines in 200 μl RPMI with 10% FBS, 2 mM L-Gln and 1 IU/ml Penicillin/ 100 µg/mL Streptomycin in above mentioned ELISPOT PVDF 96-well plates. Medium alone served as negative control. 10 μg/ml Concanavalin A (ConA, Sigma-Aldrich, St. Louis, MO) was used for general stimulation of splenocyte reactivity. 10 μg/ml MeV bulk antigens (Virion Serion, Würzburg, Germany) were used to stimulate MeV-specific T cells. After 36 h of stimulation, the splenocytes were removed from the plates, which were subsequently incubated with biotin-conjugated anti-IFN-γ antibodies and avidin-HRP according to the manufacturer´s instructions. 100 µl of 3-amino-9-ethyl-carbazole (AEC, Sigma-Aldrich) chromogen dissolved in N,N-dimethylformamide (Merck Millipore) was used for staining of spots. The reaction was stopped by washing with H₂O. Spots were counted using an Eli.Scan ELISpot Scanner (A.EL.VIS, Hamburg, Germany) and ELISpot Analysis Software (A.EL.VIS).

At endpoint, tissues were excised from uninfected Hu-mice, fixed in 10% formalin, processed, and embedded. Sections were cut at 4 µm, deparaffinized, and stained following a 5% H₂O₂ endogenous peroxidase block, antigen retrieval in citrate buffer, and blocking with 5% normal goat serum in TBS-T (20 mins). Sections were incubated with rabbit anti-human CD3 (RM-9107-S; Thermo Scientific; Waltham, Massachusetts, USA), diluted 1:100 in Ultra Clean Diluent (Fisher Scientific; Ottawa, ON) for 1 hour. Slides were rinsed in TBS-T before Dako’s Envision plus-rabbit (K4008; Dako Canada Inc; Burlington, ON) was added for 30 minutes. The slides were rinsed, and 3-Amino-9-ethylcarbazole (AEC) (Sigma-Aldrich; Oakville, ON) was employed as a chromogen (20 mins). Slides were counterstained with Mayer’s Hematoxylin (Sigma-Aldrich), 25 seconds. Sections were rinsed and coverslipped with glycerine gelatin. Images were visualized using an Olympus IX81 inverted microscope (Olympus, Richmond Hill, ON, Canada), and captured using the Infinity camera (Lumenera Corp., Ottawa, ON, Canada).