Opal 4 color manual ihc kit

Paraffin blocks of intestinal tissues derived from the AGLCD patient and 6–11 samples from different anatomical sites of 3 to 7 CD patients were cut and stained with hematoxylin and eosin (H&E) or by immunohistochemistry using antibodies listed in Supplementary Table 4. For antibody detection, the Opal 4-Color Manual IHC Kit (PerkinElmer) was used. Multispectral images were acquired using a Vectra® 3 imaging system (PerkinElmer). Positive cells were quantified in 10 high power fields (field of vision in ×400 original magnification) by inForm software (PerkinElmer). All evaluations were performed in a blinded manner. For the acquisition of immunofluorescence images depicted in Fig. 4d an AxioImager Z1 (Zeiss) was used.

Three AAmut and three AOD samples were collected for polychromatic immunofluorescence staining. Four percent formalin was used to fix the AG specimens and the specimens were embedded in paraffin blocks. For polychromatic immunofluorescence, 3 µm paraffin sections were washed in PBS twice, and permeabilized in 0.2 to 0.5% Triton X-100 (Solarbio). Then the paraffin sections were blocked in 5% normal donkey serum (Jackson Lab) and stained with primary antibody. Fluorescent-conjugated secondary antibodies (ZSGB-Bio) were used to detect the primary antibodies. Fluorescence mounting medium (Dako) was used to mount the sections. As previously described (18 (link)), we used the Opal 4-Color Manual IHC Kit (Perkin Elmer) for the analysis of formalin-fixed paraffin-embedded AG sections following the manufacturer’s protocols. Zeiss LSM880 NLO microscope was used to acquire fluorescent images. Primary antibodies were anti-CD45 (OriGene), anti-lba1 (CST), and anti-CD206 (Proteintech). GAMs were defined based on cells that costained with CD45 and lba1. CD206+ GAMs were defined based on cells that were costained with CD45, lba1, and CD206. The percentage of CD206+ GAMs was defined by (CD206+ GAMs)/GAMs.

Co-expression of amylase and pan-cytokeratin (antibodies, Supplementary Table S1) was determined using a Perkin Elmer Opal 4-color Manual IHC Kit (NEL810001KT) per the manufacturer’s instructions.

Formalin‐fixed paraffin‐embedded bone marrows were analyzed using the Opal 4‐Color Manual IHC Kit (catalog no. NEL810001KT, Perkin Elmer) per the manufacturer's instructions. A Nikon A1RMP+ multiphoton confocal microscope was used to capture the fluorescent images. The primary antibodies were anti‐CD68 (catalog no. ab222914, Abcam), anti‐iNOS (catalog no. MAB9502, R&D Systems), and anti‐ARG1 (catalog no. sc166920, Santa Cruz).

To confirm that the villin expression was independent of the subcellular localisation of PPARα, we used an Opal™ 4-Color Manual IHC Kit (Perkin Elmer, Walthem, USA, cat. no. NEL810001KT) according to the vendor’s protocol. The undifferentiated HT-29 cells were seeded in 8-well cell culture slides, adhered overnight, and treated with 150 μM fenofibrate and 10 μM GW6471 for 72 h. After that, the cells were fixed with 4% paraformaldehyde for 10 min at RT and were stained. The rabbit monoclonal primary antibody anti-villin (Abcam, Cambridge, UK, cat. no. ab130751) at a dilution of 1:100 and PPARα (GeneTex, Hsinchu, Taiwan, cat. no. GTX28934) at a dilution of 1:100 was used.

Formalin fixed paraffin-embedded specimens from 70 patients diagnosed with colorectal cancer who underwent surgical resection of tumor tissue were included in this study and analyzed by multiplex fluorescent immunohistochemistry (IHC) under an approved Institutional Review Board protocol. These patients did not receive chemoradiotherapy or immunotherapy before surgery. The primary antibodies and IHC metrics were: rabbit anti-human CCL28 (Invitrogen, 1:1500), rabbit anti-human CCR10 (Proteintech, 1:800), mouse anti-human FoxP3 (Abcam, 1:500). Multiplex fluorescent staining was obtained using Opal 4-Color Manual IHC Kit (NEL810001KT, PerkinElmer). Slides were scanned and visualized using the TissueFAXSi-plus imaging system (TissueGnostics, Vienna, Austria; acquisition software: TissueFAXS v7.0.6245) equipped with a digital Pixelink color camera (PCO AG). Multispectral images were analyzed with StrataQuest software v7.0.1.165 (TissueGnostics). The correlations of different biomarkers were evaluated by Spearman's correlation analysis (P < 0.05). Kaplan–Meier Log Rank test was used to perform univariate survival analysis. SPSS (version 25.0, IBM) and GraphPad Prism (version 8.0.1, US) were used for statistical analysis.