Pt link

After antigen retrieval (PT-Link; DakoCytomation, Glostrup, Denmark), tissue sections were subjected to automated double and triple immunostaining (Autostainer Plus; DakoCytomation). The main analysis used a triple immunohistochemistry (IHC) protocol to simultaneously detect eosinophils, basophils, and the Th2 surrogate marker GATA3. Th2 lymphocytes were identified as GATA3⁺, CD4⁺ cells. ILC2 cells were identified as lineage-negative, CD25⁺, GATA3⁺ cells after confounding non-ILC2 cells were physically blocked by prior chromogenic staining and denaturating blocking, as described previously [20 (link)]. In addition, various standard IHC cell markers were used for general immune cell exploration and for identification of chemokine-expressing cells. All antibodies had been extensively validated for use in clinical diagnosis or research (supplementary table E1).

Consecutive sections mounted on silane‐coated slides were dewaxed in xylene and hydrated through a series of graded alcohols. Next, an antigen retrieval step using a high‐ or low‐pH retrieval solution was performed in a heating instrument (PTLink, Dako) at 96°C for 20 min, following the manufacturer's instructions. After treatment with 3% hydrogen peroxide for 20 min to block endogenous peroxidase activity, the slides were incubated with a panel of primary antibodies (see Table 1) at 4°C overnight in a humidified chamber. EnVision Flex/HRP (Dako, Glostrup, Denmark) or a Vectastain ABC‐HRP Kit (Vector, USA) was used as the labeling system, following the manufacturers’ instructions, and 3,3‐diaminobenzidine tetrahydrochloride (DAB) solution (Sigma–Aldrich, Darmstadt, Germany) was used as the chromogen. The slides were counterstained with Harris hematoxylin, dehydrated and coverslipped. Photomicrographs were taken using an Olympus photomicroscope and a Nikon DS‐Fi3 camera.

TMA blocks were sectioned at a thickness of 3 μm, dried for 1 h at 65° before the pre-treatment procedure of deparaffinization, rehydration and epitope retrieval in the Pre-Treatment Module, PT-LINK (DAKO) at 95°C for 20 min in 50x Tris-EDTA buffer (DAKO, K8002), pH 9. Before staining the sections, endogenous peroxidase was blocked. The antibodies used were anti-MAP1LC3B (1:100 dilution) and anti-p62/SQSTM1 (1:100 dilution). After incubation, the reaction was visualized with the EnVisionTM FLEX Detection Kit (DAKO, K8024) using diaminobenzidine chromogen as a substrate. Sections were counterstained with hematoxylin. Appropriate negative controls including no primary antibody were also tested. The immunohistochemistry was evaluated semi-quantitatively by the intensity and the presence or not of punctate staining. To exclude subjectivity, 2 members of the team evaluated all slides independently. A histological score was obtained from each sample, which ranged from 0 (no punctate staining) to 3 (maximum immunoreactivity). Since each TMA included 2 different tumor cylinders from each case, immunohistochemical evaluation was done after examining both samples.

All sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Slides were autoclaved in citrate buffer (pH 9.0) at 97 °C for 20 min by PT Link (Dako EnVision System, Denmark). To clarify lipid accumulation, a primary antibody against adipophilin (pre-dilution, AP125; Fitzgerald Industries International, Concord, MA, United States) was used. Immunostaining for adipophilin was performed using an autoimmunostaining system (Dako EnVision System, Denmark).
The intensity of adipophilin expression was tabulated as negative, weak, moderate and strong. When the intensity of adipophilin-positive neoplastic cells was moderate or strong, the lesion was regarded as being adipophilin-positive. Depth of adipophilin expression was scored as 0 (negative), 1 (superficial expression), and 2 (deep expression) (Figure 2). When the depth of proper mucosal layer was divided into five equal layers, score 1 (superficial expression) was defined as having a vertical adipophilin expression no more than one-fifth, while score 2 (deep expression) was defined as having a vertical adipophilin expression more than one-fifth.
Scores were determined by two pathologists (ME and TS). When any difference in scoring occurred, the pathologists discussed the case until a consensus was obtained.