Pna ish detection kit

Eighteen NPC paraffin‐embedded specimens from the nasopharynx were used for immunohistochemical analysis. The patients were consecutive cases who had been diagnosed at the Division of Pathology and Otolaryngology at Kanazawa University Hospital between January 2005 and March 2012. Specimens obtained at biopsy were from 16 males and 2 females, ages 22‐80 years (mean age 60.1 years), and classified histologically as follows: 14 cases of non‐keratinizing carcinoma (type II) and 4 cases of undifferentiated carcinoma (type III) (5th TNM classification system of the International Union Against Cancer, 1997). There were 4 stage I, 6 stage III, and 8 stage IV specimens. In situ hybridization (ISH) for the detection of EBV‐encoded small RNA (EBER) was performed using the EBER PNA probe/fluorescein and PNA ISH detection kit (Dako, Glostrup, Denmark). All cases were positive for EBER‐ISH. Total DNA was isolated using TaKaRa DEXPAT Easy (Kusatsu, Japan). All samples were collected after obtaining written informed consent from the patients and were used with the approval of the Ethics Committee of Kanazawa University.

Specific infected cells together with the assessment of their histological location were determined by EBERs transcripts in situ hybridization (ISH) using PNA ISH Detection Kit (Dako), according to the manufacturer’s instructions.

Viruses used for direct infection of primary epithelial cells were concentrated by centrifugation of infectious supernatants (20,000 x g for 2 hr at 4°C) and resuspended in PBS. The viruses were applied to primary epithelial cells at a multiplicity of infection of 200 viral genomes per cell for 72 hrs. For transfer infection, primary B cells were exposed to viral supernatants at a MOI of 100 for 2 hr at room temperature and left in RPMI-20% FBS for 20hr in a CO₂ incubator [23 (link)]. Virus-loaded B cells were then washed once in culture medium used for primary epithelial cells (KGM-SFM) and cocultured with the primary epithelial cells at a concentration of 3 virus-loaded B cells per epithelial cell. The B cells were carefully removed 24 hr post-seeding and the infection rate of the primary epithelial cells was determined 48 hr thereafter by in situ hybridization with an EBER-specific PNA probe in conjunction with a PNA ISH detection kit (Dako) according to the manufacturer’s protocol. The infection experiments with the AGS cell line were performed according to the same experimental protocol, except that AGS cells were maintained in RPMI-10% FBS. We infected the cells either with unmodified viruses or with viruses trans-complemented with gp110. Infections with or without gp110 were performed in parallel.

Formalin-fixed, paraffin-embedded tissue sections were deparaffinized and then examined by IHC as previously described [95 (link)]. Antibodies used included anti-Z (BZ.1) monoclonal antibody (1:200, Santa Cruz Biotechnology SC-53904), anti-BMRF1 monoclonal antibody (1:200, Vector Laboratories VP-E608), anti-KLF4 polyclonal antibody (1:500, Sigma-Aldrich HPA002926) and anti-BLIMP1 (1:1000, Sigma-Aldrich HPA030033). Human normal tongue tissue and tonsil tissue slides were commercially purchased (Abcam and IHC World). EBER in situ hybridization studies were performed using the PNA ISH Detection Kit (DakoCytomation) according to the manufacturer’s protocol as previously described [95 (link)].

FFPE tissue sections from 10 UK lymphoma patients were tested for the presence of EBV EBER RNAs as described (Jarrett et al., 2013 (link)), using an EBV EBER PNA (peptide nucleic acid)-FITC probe and a PNA ISH Detection kit (both Dako). Samples comprised seven B-cell, two T-cell and one null-cell lymphoma; nine were from lymph node and one was from a gastric mass. The positive control was from a human EBV-associated Hodgkin lymphoma sample.

The presence of EBV was assessed by in situ hybridization for EBERs using Epstein-Barr Virus (EBER) PNA/Fluorescein (Dako, Milan-Italy), a mixture of PNA probes complementary to the two nuclear EBER RNAs encoded by EBV, in conjunction with Dako PNA ISH Detection Kit, according to manufacturer’s instructions. A control slide, prepared from a paraffin-embedded tissue block containing metastatic nasopharyngeal carcinoma in a lymph node, accompanied each hybridization run.
The expression of EBV-encoded genes (EBNA1, LMP1, EBNA2, Zebra), which characterize the different latency programs, has been investigated by qRT-PCR using Taqman probes, as previously described [21 (link)]. In addition, the expression of the relative proteins were checked by immunohistochemistry. Of these, BL cases expressed only EBNA1 at gene and protein level.