Control antibodies included Rat IgG2a isotype control mAb (eBioscience), mouse anti-Border disease virus p125/p80 mAb VPM21 and purified rabbit immunoglobulin (Sigma-Aldrich, St. Louis, MO, USA), for rat, mouse and rabbit primary antibodies, respectively. All antibodies were diluted in PBS/T80 containing 10% NGS. Slides GPCR & G Protein inhibitor were washed twice in PBS, and the appropriate secondary antibody (peroxidase-labelled anti-mouse or anti-rabbit EnVision™+ reagent, Dako) was applied to sections for 30 min at RT. After a final PBS wash, sections were incubated with 3,3′-diaminobenzidine (DAB) for 7·5 min at RT, washed in distilled water, counterstained

with haematoxylin, dehydrated and mounted in Shandon synthetic mountant (Thermo Scientific). Each nodule was scanned under the light microscope. The initial scanning was performed with a wide-angle lens at low power (×20), and the following data were recorded: the predominant inflammatory cell type, the distribution of the cell infiltrate (diffuse or focal/multifocal) and the location of the infiltrate within the nodule (peripheral, central

or both). CD3+ and Pax5+ cells tended to occur in a focal/multifocal distribution pattern in the sections, and the foci of CD3+ and Pax5+ cells were counted in the most active ×20 field (the field with the highest number of foci). CD3+ and Pax5+ infiltrates were subjectively scored 0–3 (Table 1). MAC387+ infiltrates

were also scored 0–3; however, MAC387+ cells occurred more diffusely in sections, either evenly distributed or in patches, and therefore, the scoring system was slightly different Decitabine order (Table 2). Numbers of FoxP3+ cells were counted in 10 nonoverlapping ×400 fields (five peripheral and five central fields per oesophageal nodule using a 0·0625 mm2 graticule). In the normal oesophagus control group and lymph nodes, five nonoverlapping ×400 fields were counted. Counting was confined to CD3+ areas. Statistical analyses were performed with GraphPad Prism (GraphPad Software, Inc. CA, USA). The difference in prevalence and distribution ADAMTS5 of the different proportions of cell types was tested using the chi-square test. The differences between the scores of the different types of infiltrate were tested for significance between all groups using a Kruskal–Wallis test, followed by Dunn’s post hoc test. P values of <0·05 were considered significant. Myeloid cells predominated in 70% of cases, while T cells predominated in 23% of cases. In the remaining 7% of cases, the number of T cells and myeloid cells was approximately equal. There was no difference in the proportion of myeloid and T cells between the neoplastic and non-neoplastic groups (P = 0·27). When cells were present in normal oesophageal sections, they were diffusely scattered and myeloid and T cells tended to occur in equal proportions (Table 3).