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4 6 diamidino 2 phenylindole (dapi)

Manufactured by Akoya Biosciences
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DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain used for labeling and visualizing nucleic acids, particularly DNA, in biological samples. It binds strongly to adenine-thymine (A-T) rich regions of the DNA double helix. DAPI can be used in a variety of microscopy techniques to stain the cell nuclei and facilitate the analysis of cellular and subcellular structures.

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26 protocols using 4 6 diamidino 2 phenylindole (dapi)

1

Multiplex Immunofluorescence for Immune Cell Profiling

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Using an Opal 7-color Kit (Akoya Biosciences, Marlborough, MA, USA), multiplex immunofluorescence staining was conducted on TMA sections according to the manufacturer’s protocol and as previously described by Yeong et al. (24 (link)) for simultaneous detection of CD68, myeloperoxidase (MPO), citrullinated histone H3, and DAPI. The TMA sections were baked at 65°C for 2 h and subjected to deparaffinization, rehydration, and heat-induced epitope retrieval. The sections were then incubated with a primary antibody for 1 h at room temperature, followed by incubation with an anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Akoya Biosciences). We incubated the slides with an opal fluorophore-conjugated tyramide signal amplification reagent (Akoya Biosciences). Heat-induced epitope retrieval was performed to remove the bound antibody complexes. The same procedure was repeated until all targets were detected, and the samples were labeled with DAPI (Akoya Biosciences). The following antibodies were used: anti-CD68 (D4B9C; Cell Signaling Technology, Danvers, MA, USA)/Opal 520, anti-citrullinated histone H3/Opal 620 (ab5103; Abcam, Cambridge, UK), MPO (E1E7I; Cell Signaling Technology, Danvers, MA, USA)/Opal 690. Finally, the sections were mounted using a hard-set medium.
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2

Multiplex Immunofluorescence Imaging of Tumor Microenvironment

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Multispectral imaging (MSI) was performed using the basic protocol described in Wickenhauser et al. [66 (link)]. Two multiplex panels with different mAbs and opal-dye combinations were used. The first panel included anti-PD-L1 clone E1L3N (Cell Signaling E1L3N, 1:150) in combination with Opal690, anti-Foxp3 clone 236A/E/ (Abcam, 1:00) with Opal540, anti-CD3 clone SP7 (ThermoFisher SP7, 1:100) with Opal570, anti-CD163 clone MRQ-26 (Cell Marque, 1:50) with Opal620 and anti-panCK Ab AE1/AE3 (Dako, 1:150) with Opal520. The second panel comprised anti-TIM-3 (Abcam, ab241332, 1:1000) with Opal 520, anti-PD-1 (Biocare Medical, NAT105, 1:50) with Opal 540, anti-CD8 (DAKO, C8/144b, 1:50) with Opal 570, anti-TIGIT (Biozol, USC-PAN056HU01-1, 1:50) with Opal 620, anti-CD69 (Abcam, ab233396, 1:50) with Opal 650 and anti-HLA-G (Abcam, clone 4H84, 1:100) with Opal 690. After counterstaining with DAPI (Akoya Biosciences, Marlborough, MA), the sections were mounted and scanned with the Vectra Polaris System (Akoya Biosciences, Marlborough, MA) and a mean of 18 regions of interest (ROIs) per slide were taken with a 20 × zoom. The inForm software (Version 2.4.10, Akoya Biosciences) was employed to perform cell segmentation and phenotyping. PhenoptrReports scripts were used within R to evaluate the frequency and density of the different cell types as well as their interspatial relationships.
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3

Histological Evaluation of Liver Tissue

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About 300 mg of tissue samples were taken as wedge biopsy prior to perfusion (0th hour) and at the end of perfusion (6th hour). Tissue samples were fixed in 10% neutral buffered formalin (Scigen, Gardena, CA) for 24-36 hours prior to embedding in paraffin. Sections were stained with hematoxylin and eosin for qualitative evaluation for sinusoidal and portal triad structural integrity.
Sections were also stained with anti-CD3 (BioCare medical, Pacheco, CA), CD4 (BioCare Medical), DAPI (Akoya Biosciences, Marlborough, MA) and FOXP3 (BioCare medical) (Supplemental Table 2), and slides were scanned by Vectra Polaris System (Akoya Biosciences) and analyzed with Inform Automated Image Analysis Software (Akoya Biosciences). A minimum of five regions of interest (ROI) (931 um x 698 um) were selected within each slide, in which number of tissue regulatory T cells (Treg cells) and CD3+ T cells were counted. The percentage of Treg cells out of all CD3+ T cells was calculated for each ROI.
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4

Histological and Immunohistochemical Analysis of Small Intestine

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SIs were fixed in 4% neutral-buffered formalin for 24 h and then stored in 30% sucrose until cryosections (7 μm) were cut (HM 560 Cryo-Star; Microm International GmbH, Walldorf, Germany) and subjected to routine haematoxylin and eosin staining [24 (link)].
For CD68 IHC, SIs were fixed in 4% neutral-buffered formalin for 24 h and then stored in PBS at 4 °C until paraffin embedding. Sections (7 mm) were deparaffinized, and staining was performed using a Leica Bond MAX immunostainer (Leica Microsystems, Wetzlar, Germany) and CD68 antibody (1:1,000; Cell Signaling Technologies, Danvers, MA). To visualize neutral lipids and nuclei, cryosections (7 mm) were stained with ORO (Sigma-Aldrich, St. Louis, MO) and DAPI (Akoya Bioscience, Marlborough, MA), respectively. Images were acquired at 60 × magnification (Apo 60 × Oil λS objective) using a Nikon Eclipse Ti microscope (NIKON Corporation, Tokyo, Japan) equipped with a Nikon A1 camera.
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5

Quantifying Neutrophil Infiltration in Mouse Ears

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Mice ear cryosections (6 µm) were fixed in acetone for 10 min at RT and air-dried. Then, cryosections were incubated with 400x diluted Alexa Fluor 488-conjugated rat-anti-mouse Ly-6G (GR-1 staining, eBioscience) for 1 hour at RT. After washing in PBS, nuclei were stained using DAPI (Invitrogen) at 1 µg/ml for 5 minutes at RT and embedded with fluorescence mounting medium. Tile scanning to obtain an image of the whole ear was performed using the Vectra® Polaris™ (Akoya Biosciences) microscope with the following settings: DAPI MSI 0.43ms, FITC 81.70ms and a 20 times magnification. GR-1 staining of cryosections was analyzed with ImageJ/Fiji software. The total area (µm2) of mice ears and the area of the specific GR-1 staining (µm2) was measured. Quantification was calculated as the GR-1 area (µm2) divided by the total area (µm2). The following formula was used to determine the GR-1/total area ratio:
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6

Multispectral Imaging of FFPE Samples

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Formalin-Fixed Paraffin-Embedded (FFPE) specimens from one patient both at diagnosis and recurrence were analyzed by multispectral imaging. FFPE slides were stained with anti-CD8 (clone C8/144B, Thermo Fisher Scientific, Waltham, MA, USA), anti-CD68 (clone PG-M1, Dako Agilent, Santa Clara, CA, USA) and anti-glial fibrillary acidic protein (GFAP, clone EP672Y, abcam, Cambridge, UK) antibodies for T cell, macrophage and surrounding tissue detection, respectively. DAPI (Akoya Biosciences, Marlborough, MA, USA) was used as a nuclear counterstain. The autofluorescence background signal was subtracted using an unstained control slide processed in parallel. The Mantra multispectral imaging platform (Akoya Biosciences) was employed for image acquisition at 20× magnification and data analysis was carried out with InForm 2.4.1 software (Akoya Biosciences). The immune cell infiltrate was quantified as cell density/megapixel.
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7

Multiplexed Immunohistochemistry for Tissue Analysis

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A multiplexed immunohistochemistry (mIHC) analysis was performed on formalin‐fixed paraffin‐embedded (FFPE) tissue slides using the Mantra multispectral imaging platform (Akoya Biosciences, Marlborough, Massachusetts). FFPE slides were stained with anti‐CD8 (clone C8/144B, Thermo Fisher Scientific), anti‐CD68 (clone PG‐M1, Dako Agilent, Santa Clara, California), anti‐HO‐1 (clone HO‐1‐1, Thermo Fisher Scientific) and anti‐glial fibrillary acidic protein (GFAP, clone EP672Y, Abcam, Cambridge, UK) antibodies. DAPI (Akoya Biosciences) was used as a nuclear counterstain. The autofluorescence background signal was subtracted by using an unstained control slide processed in parallel. The Mantra multispectral imaging platform (Akoya Biosciences) was employed for image acquisition at ×20 magnification and data analysis was carried out with InForm 2.4.1 software (Akoya Biosciences).
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8

Multiplex Immunohistochemistry Staining Protocol

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Opal™ 7-Color Multiplex IHC Kit (Akoya Biosciences, NEL861001KT) was employed to perform multiplex staining. The protocol was referred to the manufacturer’s construction. FFPE sections were incubated at 65°C for at least 18 h as preprocessing. The slide underwent a serial deparaffinization step and then immersed to quench peroxidase followed by washing. The following steps were repeated for multiple-marker staining. The slides were successively treated for primary antigen retrieval, blocking of unspecific binding, secondary antibody conjugating, and stripping. After completing multiple stainings, the slides were scanned on the PerkinElmer Vectra 3® Polaris™ platform and imaged on the inForm Advanced Image Analysis software (inForm 2.4.1; Akoya Biosciences, USA) with the DAPI (Akoya Biosciences) filter set. The antibodies and reagents are listed in Table S1.
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9

Multiplex Immunofluorescence for NSCLC Immune Profiling

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Two mIF panels were developed to characterize the locally advanced NSCLC immune microenvironment. CD4 panel including Pan-CK, CD4, FoxP3, CD8, CD31, α-SMA, HIF-1α and DAPI. CD8 panel including Pan-CK, CD8, CD103, PD-1, TIM-3 and DAPI.
The procedure for mIF has been previously described (21 (link), 30 (link)–33 (link)). In summary, 3 μm serial sections obtained from TMA blocks were deparaffinized and rehydrated using a decreasing ethanol series. Antigen dretrieval was performed by using microwaves. A blocking buffer was then used to initiate protein stabilization and background reduction for 30-60 minutes at room temperature. Primary antibodies were applied and washed in 1×Tris-buffered saline containing 0.5% Tween 20. Isotype-specific HRP-conjugated antibodies and an Opal fluorophore (Akoya Biosciences). Finally, the nuclei were stained with DAPI (Akoya Biosciences) for 10 min. Antibodies and fluorophores used in the mIF procedures were detailed in Supplementary Table 1.
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10

Histological analysis of small intestine

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SIs were fixed in 4% neutral-buffered formalin for 24 h and then stored in 30% sucrose until cryosections (7 μm) were cut (HM 560 Cryo-Star; Microm International GmbH, Walldorf, Germany) and subjected to routine haematoxylin and eosin staining [24 (link)].
For CD68 IHC, SIs were fixed in 4% neutral-buffered formalin for 24 h and then stored in PBS at 4 °C until paraffin embedding. Sections (7 μm) were deparaffinized, and staining was performed using a Leica Bond MAX immunostainer (Leica Microsystems, Wetzlar, Germany) and CD68 antibody (1:1,000; Cell Signaling Technologies, Danvers, MA). To visualize neutral lipids and nuclei, cryosections (7 μm) were stained with ORO (Sigma-Aldrich, St. Louis, MO) and DAPI (Akoya Bioscience, Marlborough, MA), respectively. Images were acquired at 60 × magnification (Apo 60× Oil λS objective) using a Nikon Eclipse Ti microscope (NIKON Corporation, Tokyo, Japan) equipped with a Nikon A1 camera.
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