Aperio at turbo

Antigen retrieval for staining was for 20 min at 100 °C in a 1 mM Tris, 1 mM EDTA, pH 9 buffer. BRN2 (12137 S; CST) or MITF (D5G7V; CST) antibodies were diluted 1:100 in Da Vinci Green (Biocare Medical, Concord, CA) and incubated overnight at room temperature. Sections were then incubated in MACH 1 Universal HRP polymer (Biocare Medical) for 30 min, counterstained with Nova Red (DakoCytomation, Glostrup, Denmark) followed by haematoxylin. Additional sections were also stained with haematoxylin and eosin following standard procedures. All stained sections were scanned with the Aperio AT Turbo (Leica Biosystems, Nussloch, Germany) and images extracted in ImageScope v11 (Leica Biosystems). Total tumor area per lung was determined by staining fully serial sectioning formalin-fixed, paraffin-embedded lungs by IHC with a BRN2 antibody. After scanning with the Aperio AT Turbo the Genie macro for ImageScope (Leica Biosystems) was used to measure the total tumor area and total lung area per section.

All stained sections were scanned by the Aperio AT Turbo (Leica Microsystems). ImageScope 12.1(Leica Microsystems) was used to select 9 consecutive square areas for each case. The area of each small square area was fixed to 0.09 mm² and the same selected areas were procured for assessment in the serial sections (Fig. 1). Counting eosinophil was performed by experienced pathologists.Figure 1

Select the same area for quantitative assessment (×50).

All of the statistical analyses were performed using the GraphPad prism 5. The counting data were not normally distributed showed by the Shapiro-Wilk’s test. Therefore, the Friedman test was performed to determine whether there were significant differences among the conventional HE, Chromotrope 2R, Congo red and MBPmAb IHC for the eosinophil counting data. The Wilcoxon signed-rank test was used for the pairwise comparisons, if the significant differences were noted. The Bonferroni correction was applied in pairwise comparisons and the adjusted p = 0.05/6 was considered significant. All tests were done using the two-tailed option. For all analyses, P < 0.05 was considered significant.

Harvested tissues were fixed in 4% paraformaldehyde (PFA) overnight and embedded in paraffin. All specimens were cut to 4‐μm‐thick sections and subjected to hematoxylin and eosin (H&E), ISH, and IHC (Ab information in Appendix Table S5). IHC and quantification of macrophages and αSMA expression were performed as previously described (Mori et al, 2006, 2008, 2014). Observations were made via digital whole slide scanning system (Aperio AT Turbo; Leica Microsystems, Tokyo, Japan) confocal microscopy (C2+ system; Nikon Corp., Tokyo, Japan)]. Aperio eSlide Manager (Leica Microsystems), NIS‐Elements C software version 4.13 (Nikon Corp.), AR software version 4.0 (Nikon Corp.), or IMARIS 7.7.2 (Bitplane, Zurich, Switzerland) were used for data analysis.