Envision plus kit

A CXCL13 kit was bought from R&D Company in the United States. Other reagents included Clone PS1, Novacastra as a CD3 antibody (T-cell surface marker); Clone L26, DAKO Company as a CD20 antibody (B-cell surface marker); and Clone 2G9, Gene Company as a CD21 antibody [follicular dendritic cell (FDC) surface marker]. DAKO EnVision plus kit was used for immunohistochemical test. MN1616, German BARD Company, was used as a percutaneous automatic biopsy gun.

Immunostaining was performed using an EnVision Plus kit (Dako, Glostrup, Denmark). We cut 4 μm sections from the microarray tissue. After dewaxing and hydration, they were placed in a bath of hot citrate buffer, pH 6.0 at 95 °C for 40 min for BC-1514 (C15), BC-1514 (W12), CAMK2N1, CD1D, PJA2, SAMD13, TCF4 and TXNIP, or of Target Retrieval Solution, pH 9.0 (Dako) for RPL5 at 95 °C for 40 min. The sections were incubated with the primary antibodies for overnight at 4 °C. The dilutions of primary antibodies were: 1:200 for BC-1514 (C15), 1:100 for BC-1514 (W12), 1:500 for CAMK2N1, 1:300 for CD1D, 1:100 for PJA2, and 1:1000 for RPL5. For SAMD13, TCF4, and TXNIP, the antibodies were used at 2 μg/ml. The chromogen used was 3,3′ -diaminobenzidine tetrachloride, and the sections were counterstained with hematoxylin.
Immunohistochemistry was analysed using a histoscore that was calculated by multiplying the positive area (%) and intensity (0–3: 0 for negative, 1 for weak, 2 for moderate, and 3 for strong staining). Immunohistochemical analyses were evaluated in blinded manner.

Portions of brain and lung of PCR positive animals, previously fixed in formalin, were embedded in paraffin, cut at thickness of 5 µm and routinely stained with hematoxylin and eosin.
Immunohistochemistry was carried out on newly prepared unstained sections of both organs, using the following procedure: blocking of endogenous peroxidase with 3% H₂O₂ at room temperature for 30 min, antigen retrieving with trypsin at 37 °C for 30 min, overnight incubation at 4 °C with a monoclonal antibody anti-Canine Distemper Virus (clone 8-1) (LSBio). A positive reaction was detected using 3, 3′- diaminobenzidine (EnVision Plus kit, Dako) as chromogen with a 3-min development at room temperature and counterstained with hematoxylin.

Optimal tissue blocks were selected by an expert pathologist on haematoxylin and eosin (H&E) slides. For each patient, representative areas of normal tissue, tumor tissue, preneoplastic lesions grade 2–3 (PanIN), and lymph node metastases were selected and included in tissue microarrays (TMA). For each sample, two representative cores of 1.2 mm in diameter were arrayed into a receptor block using a TMA workstation (Beecher Instruments, Silver Spring, MD, USA) as previously described [16 (link)]. For IHC assays, 4 µm sections of the TMAs were used. Slides were deparaffinized and rehydrated. Antigen retrieval was performed in a DAKO PT Link, incubated with specific primary antibodies: GATA-4 clone 532020 ref. n° MAB2606 and GATA-6 ref. n° AF1700 (R&D Systems, MN, USA), detected with a Dako Envision Plus kit, and counterstained with haematoxylin. All reagents are from Dako (Agilent, CA, USA). The staining was assessed by two independent pathologists, blinded to the origin of the samples. Both the intensity of the staining (0—negative, 1—weak, and 2—strong) and the percentage of positive cells of each tissue core were determined. An H-score (intensity x% positive cells) was calculated, ranging from 0 to 300). H-scores of <30 were considered negative [17 (link)].