Real detection system

Initially, two brain regions per animal were screened by IHC, with samples including the hippocampus, obex, or cerebellum being preferentially used. Subsequently, all brain regions available for the positive case were investigated. The immunohistochemical staining procedure was essentially the same as the one we described previously (17 (link)). Two kinds of polyclonal antibodies were obtained by immunizing rabbits with recombinant viral proteins (that were designed on the capsid protein sequence of BoAstV-CH15). IHC was performed as follows: tissue sections were deparaffinized, rehydrated, and endogenous peroxidase activity was blocked in a solution of 3% H₂O₂ in methanol. Subsequently, they were microwave cooked in Dako Target Retrieval Solution, Citrate pH 6 (Agilent) and blocked with 10% normal goat serum in PBS-T. The samples were incubated with the primary antibody CH15-ORF2-var (diluted 1:50 in PBS-T) and detection was carried out with Dako REAL Detection System, following the manufacturer's instructions. For the positive case, all brain regions were also tested with the primary antibody CH15-ORF2-con (17 (link)), using the same procedure as above. All samples were run in parallel with OvAstV-CH16-positive and -negative controls.

Immunohistochemical detection of VRK1 in patient samples was performed using formalin-fixed and paraffin-embedded tissue sections (∼4 μm thick). After deparaffinization in xylene for 15 min, rehydrated tissue sections were subjected to antigen retrieval by boiling in Tris-EDTA buffer (pH 9.0) for 5 min. Slides were then incubated with rabbit anti-VRK1 (Santa Cruz) for 1 h at room temperature, and the VRK1 antigen-antibody reaction was detected using a Real Detection System (Dako, K5001). Immunohistochemical intensity of VRK1 staining was evaluated by two independent pathologists. VRK1 expression was evaluated in 10 high-power fields (400 ×). In each sample, intensity was classified as 0 (negative staining), 1 (<5% staining), 2 (<25% staining), 3 (25–50% staining) and 4 (>50% staining), and the average intensity assessed. Immunohistochemical detection of Ki-67 in mouse tumors treated with luteolin was also performed using the same method described above with mouse anti-Ki-67 (Dako, M7240) antibody.

IHC was carried out following a recently published protocol [26 ]. Four µm-thick sections were mounted on positive charged glass slides, dried overnight at 37 °C, dewaxed and rehydrated using standard procedures. Antigen retrieval was performed by incubation with trypsin (Sigma-Aldrich; working solution = 0.01% in 0.15 M Tris–HCl buffer, pH 7.8) at 37 °C for 10 min. A murine monoclonal antibody anti-Mmm was used as primary antibody. Immune reactions were detected by means of a biotin-streptavidin amplification method and visualized using 3-3′-diaminobenzidine as chromogen (Dako REAL™ detection system). Lung samples from CBPP-naturally affected cattle which showed lesions at different stages of evolution (red-to-grey hepatization, necrosis, sequestra) acted as positive control. Negative controls consisted of CBPP negative lung samples and were also included in each IHC run.
DLIIF and LSCM investigations were carried out to better detail the cellular tropism of Mmm, both in challenged BREs and in samples collected from CBPP-affected cattle. To this purpose, tissue sections were incubated with primary antibodies anti-Mmm, lysozyme, von Willebrand factor (vWF) and cytokeratins (see Additional file 3 for details). Sections were mounted using a antifade medium with DAPI (Vector Laboratories, Inc.), stored at 4 °C in the dark until imaged using a Leica TCS SP5 II confocal microscope.

In brief: For fetuin-A staining, we used a monoclonal IgG2a mouse-anti-human antibody (clone MAHS-1, dilution 0.1–0.5 μg/mL), raised against purified human fetuin-A in our laboratories. Antibody specificity had been confirmed by Western blotting [29 (link)]. Antibodies were diluted in a 1% dilution of Bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and were immediately applied to the re-hydrated sections. Bound antibody was detected using Dako REAL Detection System, which employs APAAP immunochemistry and fast red chromogenic substrate (Dako K5000, Glostrup, Denmark) following the manufacturers protocol. Counterstaining was employed with Mayer´s hematoxylin solution (Roth, T160.1, one minute). The slides were then washed in demineralized water and dehydrated in graded alcohol (concentrations from 70% to 100%). After placing in xylene, the sections were mounted (Roth, T160.1) and covered using coverslips. For details see http://dx.doi.org/10.17504/protocols.io.sydefs6 [PROTOCOL DOI].

Sections from lymph nodal biopsies and prostate cancer biopsies were incubated with mouse monoclonal Anti-Endothelin1 antibody (Calbiochem, Merck, Darmstadt, Germany, dilution 1∶250) or with rabbit polyclonal ET_AR antibody (Abgent, dilution 1∶50). Detection of bound antibody was performed with alkaline phosphatase method using a streptavidin-biotin alkaline phosphatase complex kit (REAL Detection System, Dako, Glostrup, Denmark). The alkaline phosphatase reaction was then developed with Permanent Red (Dako) as chromogen.

Sagittal sections were performed at 4 μm thickness from paraffin blocks of enucleated eyes from in vivo tumor models (n = 6) and human patients (n = 6). Immunohistochemical analysis with human eyes was approved by Institutional Review Board of Seoul National University Hospital (H-1404-032-568) and clinical demographics of 6 patients were summarized in Supplemental Table 1. We also followed Declaration of Helsinki during the whole procedures with human tissue samples. The sections were incubated at 60°C for 2 hours and processed with deparaffinization and hydrated by sequential immersion in Xylene Substitute (Thermo) and graded ethyl alcohol solutions. Antigen retrieval was performed by the treatment with 0.1 M sodium citrate (pH 6.8, Sigma) at 120°C for 10 minutes. The sections were permeabilized with 0.2% Triton X-100 at room temperature for 10 minutes. Then, we treated the sections with 1X Universal Blocking Reagent (Biogenex) for 10 minutes to minimize nonspecific binding. After incubation with primary antibodies (1:250) overnight, the sections were treated with REAL™ Detection Systems (Dako) and DAB Kit (Life Technologies) as the manufacturer's instructions. Then, the slides were mounted with Permount solution (Thermo) and observed under the light microscope (Nikon).

From paraffin blocks of enucleated eyes from 30 patients, sagittal sections were prepared at 4 μm thickness. The sections were incubated at 60°C for 2 hours and then deparaffinized and hydrated by sequential immersion in Xylene Substitute (Thermo) and graded ethyl alcohol solutions. Antigen retrieval was performed by the treatment with 0.1M sodium citrate (pH 6.8, Sigma) at 120°C for 10 minutes. The sections were permeabilized with 0.2% Triton X-100 at room temperature for 10 minutes. Then, to minimize nonspecific binding, the sections were treated with 1X Universal Blocking Reagent (Biogenex) for 10 minutes. After incubation with the primary antibody against L1 (1:5000; cat. no. ab24345, abcam) overnight, the sections were treated with REAL™ Detection Systems (Dako) and DAB Kit (Life Technologies) according to the manufacturer's instructions. Then, the sections were mounted with Permount solution (Thermo) and observed under the light microscope (Nikon). The percentage of L1-positive cells in tumor sections were evaluated by an experienced pathologist (Y.H. Kim) and confirmed by another independent observer (D.H. Jo). In addition, the number of Flexner-Wintersteiner rosettes in tumor samples were estimated from direct counting throughout the whole tumor sections.