M3515

IHC based on MAC387 (Serotec MCA874G, diluted 1:1000), staining of macrophages and NGs, and cytokeratin staining (Mouse anti-cytokeratin clone AE1/AE3, DAKO M3515, diluted 1:1200) were used as previously described [26 (link)–28 (link)] on various intestinal sections from selected pigs with crypt abscesses in order to confirm the composition of the debris.

Immunohistochemistry was performed as previously described [41 (link)]. Tissue sections were blocked with Background Sniper (BS966, Biocare Medical). Antigen Retrieval was performed in Tris/EDTA pH 9 Buffer for 20 minutes (S2367, Dako). The sections were incubated overnight with the appropriate primary antibody: p^T356RB1 (1:200, 2223-1, Epitomics), RB1 (1:200, #9309, Cell Signaling Technology), p^T202/Y204ERK1/2 (1:100, #9101S, Cell Signaling Technology), N-terminal TP53 (1:200, M7001, DAKO), or Ki-67 (1:800, AC-0009, Epitomics), and pan-cytokeratin (Rabbit 1:400, Z0622, Dako or Mouse, 1:100, M3515, Dako; tumor mask) in Da Vinci Green antibody diluent (PD900, Biocare Medical) at 4°C overnight. Signals were intensified with Envision reagents (DAKO). Pan-cytokeratin primary antibody was probed with an Alexa Fluor 555 dye-labeled secondary antibody (Invitrogen). Primary antibody visualization was accomplished using a Cy-5-tyramide signal amplification system (TSA; AT705A, PerkinElmer). Tissue nuclei were stained using Prolong Gold mounting medium (P36931; Molecular Probes) containing 4,6-diamidino-2-phenylindole (DAPI). HistoRx PM-2000 (HistoRx) with AQUAsition software was used for automated image capture as previously described [41 (link)].

Three 4-µm sections (n ​= 246) were cut and placed on a platinum-coated slide of Dako (Denmark, Glostrup, K8020). Pretreatment methods were performed using Dako’s PT link. Using the heat-induced targeting solution of Dako (EnVision Flex), the retrieval epitope was obtained at pH 9, 97 ° C for 20 min. The staining was performed using Dako’s Autostainer link 48. Endogenous peroxidase activity was blocked by Dako’s peroxidase blocking reagent (EnVision Flex). The primary antibody was mouse monoclonal AE1/AE3 (Dako, clone M3515, 1:250) diluted with the antibody diluent of Dako (EnVision Flex). IHC staining of mismatch repair proteins was performed using mouse monoclonal MLH1 (Dako, clone ES05, 1:100) and PMS2 (Dako, clone A16-4, 1:500) antibodies. These antibodies were incubated for 30 min at room temperature and the mouse linker of Dako (EnVision Flex) was used for amplification. The bound antibody was detected by HRP reaction of Dako (EnVision Flex) and visualized by DAB reaction of Dako (EnVision Flex). Meyer hematoxylin (Merck, Germany, Darmstadt) was used for counterstaining and Pertex (Histolab, Sweden, Gothenburg) was used to cover the slides.

An immunofluorescence assay was performed to detect the expression of the α-SMA (1:200, mouse anti-alpha-smooth muscle actin, M0851, Dako, USA), Vimentin (1:200, mouse anti-vimentin, M0725, Dako, USA), Cytokeratin (1:200,mouse anti-cytokeratin, M3515, Dako, USA) in the MGT cell lines. MGT cell lines were seeded in 24-well plates with placed 12 mm coverslips (SPL, Korea). Cells were fixed in 4% paraformaldehyde solution for 15 min at room temperature, permeabilized in 0.2% Triton X-100 (9036-19-5, Sigma-Aldrich) for 10 min, and blocked with 5% bovine serum albumin (BSA) (9048-46-8, Sigma-Aldrich) in D-PBS for 1 h. The cells were incubated with primary antibodies diluted in D-PBS (1:200) with 5% BSA overnight at 4 °C, then incubated with the secondary antibody (1:400) for 2 h at 37℃. Each stage was washed three times for 5 min with D-PBS. All coverslips were mounted with a mounting solution with DAPI (101098-044, Vector laboratories, CA, USA). Slides were imaged with confocal microscopy (Carl Zeiss, Germany).

TTF-1 and CK7 IHC was carried out on formalin-fixed, paraffin-embedded tissue 4 μm sections using anti-TTF-1 (mouse, 8GTG3/1, 1:200, Dako, Carpinteria, CA, USA) and anti-CK7 (mouse, OV-TL 12/30, 1:1000, Dako). Antibody incubation and detection were carried out at 37°C on a Menarini intelliPATH FLX (A. Menarini Diagnostics, UK) using Menarini's reagent buffer and detection kits unless otherwise noted. Antigen retrieval was carried out in a pressure cooker in citrate buffer pH6 for 2 min at 123°C. Pan-CK, CD44 and vimentin IHC was carried out on formalin-fixed, paraffin-embedded tissue 4 μm sections using anti pan-cytokeratin antibody (mouse, M3515, 1:60, ER1 20 min Dako), anti CD44 (mouse, DF1485, 1:100, ER2 10 min, Dako) and anti-Vimentin (mouse, V9, CC1 32 min, Roche). Pan-CK and CD44 staining was carried out on the LEICA Bond Max Platform and Vimentin staining on the Ventana Discovery Ultra platform (Roche). Appropriate positive, negative and isotype controls were included with the study sections (not shown). Digital images of whole-tissue sections acquired using a Leica SCN400 histology scanner (Leica Microsystems).

Formalin‐fixed paraffin‐embedded (FFPE) tissue from lymph nodes was sectioned (4 µm) and stained for hematoxylin and eosin (HE) and IHC‐stained for CD4 (4B12, Thermo Fisher), CD8 (NCL‐CD8‐4B11, Leica, Leica Biosystems, Wetzlar, Germany), and AE1/AE3 (cytokeratin CK1‐8, 10, 14‐16 and 19, M3515, DAKO, GmbH, Jena, Germany). Immunofluorescence was performed with high PH (Envision ™ FLEX target retrieval solution, DM828, DAKO) antigen retrieval and stained with CD8 ((C8/144B, cat.nr: 372902 BioLegend, San Diego, CA, USA)+ biotin (Gt anti‐mouse, M30115, Invitrogen, Thermo Fisher Scientific, MA, USA)+ streptavidin‐conjugated AF488 (S11223, Invitrogen)) and FoxP3 (236A/E7,ab20034 Abcam + goat‐anti‐Mouse AF594 (A21125, Invitrogen)) and counterstained with DAPI (Invitrogen™ ProLong™ Gold Antifade Mountant with DAPI, P36941, Fisher Scientific). The stainings were performed according to manufacture procedures. The sections were photographed in a Zeiss Axiovision Imager M1 microscope equipped with Axiocam MRc and Axiocam MRrm.