Livers containing islet grafts were procured from recipient mice, fixed in 10% formalin neutral buffer solution, embedded in paraffin, and cut into 5 μm-thick sections. Hematoxylin and eosin (H&E) staining was preformed according to standard protocols. Immunohistochemical staining was performed as previously reported.25 (link) Sections were incubated overnight at 4°C with the following primary antibodies: antiinsulin (1:2000; Proteintech, Tokyo, Japan), anti-CD4 (1:100; D7D2Z, Cell Signaling Technology), anti-CD8α (1:400; D4W2Z, Cell Signaling Technology), anti-CD31 (1:100; Cell Signaling Technology), and anti-F4/80 (1:50; Novus Biologicals), followed by incubation for 40 min with a biotinylated secondary antibody diluted 1:300 in PBS. Sections were washed with PBS before addition of avidin-biotin-peroxidase complex (1:100 in biotinylated secondary antibody; ABC-Elite, Vector Laboratories, Burlingame, CA) for 50 min. After washing in PBS, the color reaction was carried out using diaminobenzidine and nuclei were counterstained with hematoxylin. The number of positively stained lymphocytes that had infiltrated into the islet grafts was counted in all lobes of the liver (at the maximum cross section, which contained about 10 islets in total). The areas of liver sections that stained positively for insulin were measured and analyzed using NIH Image J software.
D4w2z
Manufactured by Cell Signaling Technology
Sourced in United States
D4W2Z is a specialized laboratory equipment designed for cell culture applications. It functions as an incubator, providing a controlled environment for the growth and maintenance of cell lines. The device regulates temperature, humidity, and gas composition to support optimal cell culture conditions.
Automatically generated - may contain errors
Lab products found in correlation
D7d2z, by Cell Signaling Technology (3 mentions)
Bond rx, by Leica (2 mentions)
Halo software, by Indica Labs (2 mentions)
Cellsens, by Olympus (2 mentions)
Dp80 microscope, by Olympus (2 mentions)
Epr19514, by Abcam (2 mentions)
Abc elite, by Vector Laboratories (1 mentions)
Anti insulin, by Proteintech (1 mentions)
Anti cd31, by Cell Signaling Technology (1 mentions)
Anti f4 80, by Novus Biologicals (1 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Immpress anti rat ig, by Vector Laboratories (1 mentions)
Immpress anti rabbit ig, by Vector Laboratories (1 mentions)
Plan apochromat 20 0.8 objective, by 3DHISTECH (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
Prolong gold mounting medium with dapi, by Thermo Fisher Scientific (1 mentions)
Citrate solution, by Agilent Technologies (1 mentions)
Goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Appropriate serum, by Agilent Technologies (1 mentions)
Pa5 114995, by Thermo Fisher Scientific (1 mentions)
16 protocols using d4w2z
1
Histological Analysis of Islet Grafts in Liver
2
Immunohistochemical Analysis of Liver Tumor
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Paraffin-embedded liver/tumor
tissue sections were deparaffinized, rehydrated, underwent heat-induced
antigen retrieval, and then incubated with primary Abs. The primary
Abs types used for detection of Ki-67, F4/80, Gr-1, and CD8alpha were
SP6 (Abcam), BM8 (Biolegend), RB6F8C5 (Biolegend), and D4W2Z (Cell
Signaling Technology), respectively. ImmPRESS anti-rat Ig, ImmPRESS
anti-rabbit Ig, polymer detection kits, DAB peroxidase substrate kit,
(Vector laboratories) liquid permanent red substrate (Dako), and Hematoxylin
Gill II (Leica) were used for detection and visualization. The images
were captured using an automatic digital slide scanner Pannoramic
MIDI with a Plan-Apochromat 20×/0.8 objective (3D HISTECH) by
the Pathology Core Laboratory of NHRI.
tissue sections were deparaffinized, rehydrated, underwent heat-induced
antigen retrieval, and then incubated with primary Abs. The primary
Abs types used for detection of Ki-67, F4/80, Gr-1, and CD8alpha were
SP6 (Abcam), BM8 (Biolegend), RB6F8C5 (Biolegend), and D4W2Z (Cell
Signaling Technology), respectively. ImmPRESS anti-rat Ig, ImmPRESS
anti-rabbit Ig, polymer detection kits, DAB peroxidase substrate kit,
(Vector laboratories) liquid permanent red substrate (Dako), and Hematoxylin
Gill II (Leica) were used for detection and visualization. The images
were captured using an automatic digital slide scanner Pannoramic
MIDI with a Plan-Apochromat 20×/0.8 objective (3D HISTECH) by
the Pathology Core Laboratory of NHRI.
3
Immunohistochemical Staining for CD8+ T Cells
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For serial sections cut at 7-μm thickness, tissue samples were fixed in 2% paraformaldehyde overnight at 4°C, dehydrated, and embedded in paraffin. Paraffin slides were first rehydrated to proceed with antigen retrieval in citrate solution (Dako). Subsequently, the sections were blocked with the appropriate serum (Dako) and incubated overnight with the rabbit anti-CD8 antibody (D4W2Z; Cell Signaling Technology) at 1:200 dilution. Next, the secondary goat anti-rabbit antibody (Jackson Immunoresearch, West Grove, PA) was used at 1:300 dilution. Whenever sections were stained in fluorescence, ProLong Gold mounting medium with DAPI (Invitrogen, San Diego, CA) was used. Microscopic analysis was done with an Olympus BX41 microscope (Olympus, Tokyo, Japan) and CellSense imaging software V2.3 (Olympus Lifescience, Tokyo, Japan).
4
Immunohistochemical Analysis of Immune Markers
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Immunohistochemistry (IHC) was performed using a CD86 (1:500, tris, PA5-114995, ThermoFisher), Arg1 (1:1000, citrate, 16001-1-AP, Proteintech), and CD8 antibodies (1:400, tris, D4W2Z, Cell Signaling Tech). Tumors were harvested, formalin fixed, embedded in paraffin, and sectioned at 5 µm. After deparaffinization in xylene and graded EtOH, antigen retrieval was performed using Tris buffer at pH 9 (CD86, CD8) or Citrate at pH 4 (Arg1). Primary antibody incubation was performed overnight at 4°C in a humidified chamber. Secondary antibody incubations and detection via alkaline phosphatase per manufacturer instruction (Vector Red AP Kit, SK-5100). Cell enumeration was conducted using Trainable Weka Segmentation, available through the Fiji image processing distribution of ImageJ (26 (link), 27 (link)).
5
Quantitative Analysis of CD8+ and CD11c+ Cells in Tumor Specimens
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Formalin-fixed paraffin-embedded (FFPE) specimens from human clinical samples were cut into 4 µm sections and mounted on glass slides. Antigen retrieval was performed in citrate buffer (pH 6) at 121 °C for 20 min. CD8+ cell infiltration within the tumour gland was observed and blindly scored by an experienced pathologist according to the following criteria: 0 (0–5% CD8 staining); 1 (5–10% CD8 staining); 2 (10–15% CD8 staining); and 3 (>15% CD8 staining). Simultaneously, CD8+ cells were quantified using an automated imaging system (Vectra 3.0; Perkin-Elmer, Waltham, MA, USA) equipped with the analytical software InForm 2.2 (Perkin-Elmer).
Frozen tissue specimens from mice were cut into 6 µm sections and mounted on glass slides. Individual slides were incubated overnight at 4 °C with anti-CD11c mAb (D1V9Y; Cell Signalling Technology, Danvers, MA) and anti-CD8 mAb (D4W2Z; Cell Signaling Technology). The slides were then incubated in SignalStain® Boost IHC Detection Reagent (Cell Signaling Technology), and the colour was developed using a Signalstain® DAB Substrate Kit (Cell Signalling Technology). CD11c- and CD8-positive cells were counted; three fields (×400) comprising tumour cells were randomly selected and counted for each slide. The mean of the three area counts for each tumour was used for statistical analysis.
Frozen tissue specimens from mice were cut into 6 µm sections and mounted on glass slides. Individual slides were incubated overnight at 4 °C with anti-CD11c mAb (D1V9Y; Cell Signalling Technology, Danvers, MA) and anti-CD8 mAb (D4W2Z; Cell Signaling Technology). The slides were then incubated in SignalStain® Boost IHC Detection Reagent (Cell Signaling Technology), and the colour was developed using a Signalstain® DAB Substrate Kit (Cell Signalling Technology). CD11c- and CD8-positive cells were counted; three fields (×400) comprising tumour cells were randomly selected and counted for each slide. The mean of the three area counts for each tumour was used for statistical analysis.
6
Quantifying T-Cell Infiltration in Murine Tumors
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To quantitatively measure the increase in CD8+ T cells and CD3+ T cells after YH29407 + aPD-1 treatment, mouse tumor tissues obtained through internal sacrifice were sectioned into five sheets each after formalin-fixed paraffin-embedded preservation and analyzed by IHC. Fluorescence data were re-implemented mixed images of the 3,3′-Diaminobenzidine result through multispectral image analysis, and the CD3+ and CD8+ cells were stained yellow. All tumor tissue was analyzed, and 15 to 40 fields were analyzed per sample. The results were derived by quantifying the total number of cells and the number of CD3+ and CD8+ cells per mm2. Whole slide scanning and cell segmentation were performed to quantify the IHC results, and the degree of CD8+ and CD3+ T lymphocyte infiltration was measured using Vectra Polaris and InForm software. IHC was performed using an automatic staining machine (LEICA BOND RX). Digital images of IHC slides were obtained using a whole slide scanner. Image deconvolution was performed using InForm software. The slides were stained with CD3e (CD3-12, Cell Signaling Technology, Beverly, MA, USA) and CD8α (D4W2Z, Cell Signaling Technology).
7
Quantifying CD8+ T Cells in Brain Tumors
8
Quantifying CD4+ and CD8+ T Cells
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Formalin-fixed paraffin embedded tissues were cut into 4 µm sections and stained with anti-CD4 (1:100, D7D2Z, Cell Signaling Technology, Danvers, Massachusetts, USA) or anti-CD8 mAb (1:400, D4W2Z, Cell Signaling) by the HRP method, followed by standard chromogenic immunohistochemistry protocol. A detailed method is shown in online supplemental methods . Stained slides were scanned on a DP80 microscope (OLYMPUS, Tokyo, Japan) and digital images were viewed using cellSens (OLYMPUS). CD4+T cells and CD8+T cells were counted in randomly selected five different high-power fields to obtain an average number for each condition.
9
Immunohistochemical Quantification of CD8+ Cells
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IHC was performed as we have previously described [41 (link)]. Deparaffinization of slides was carried out in xylene, followed by ethanol clearance and antigen retrieval by means of heat treatment in a sodium citrate buffer (pH = 6.0). Non-specific binding sites were blocked with PBS containing 1% BSA and 10% normal goat serum (Vector Laboratories, Newark, CA, USA) for 1 h, followed by the addition of the anti-CD8 primary antibody (Cell Signaling #D4W2Z, 1:100) overnight at 4 °C and biotinylated goat anti-rabbit IgG for 1 h at room temperature. Chromogenic reaction (Vector Laboratories) and slide counterstaining with hematoxylin (Sigma Aldrich, St. Louis, MI, USA) were then carried out. Images were captured and analyzed with ImageScope software (Leica Microsystems Inc, Wetzlar, Germany); quantification was performed by counting the total number of cells and CD8+ cells; and CD8+ cells were expressed as the % of CD8+ cells. Non-specific IgG was used as a negative control. IHC images were also downloaded from the Human Protein Atlas (https://www.proteinatlas.org/ ) (accessed on 27 December 2022) using the HPAanalyze R package [42 (link)].
10
Histological and Immunofluorescence Analysis of Mouse Tissues
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Mouse tissues were fixed in 10% buffered formalin (Fisher Scientific, catalog SF100-20) overnight and embedded in paraffin. Sections (8 μm thick) were stained with H&E. Images were acquired on a TissueFAXS Whole Slide Scanning System (TissueGnostics) using a 20x objective. Nuclei quantification was performed with Image J.
For immunofluorescence staining, muscle sections were de-paraffinized in xylene and rehydrated using a graded ethanol series culminating with PBS. Following antigen retrieval using a programmable pressure cooker with “target retrieval solution”, pH 6.0 (Dako), tissue sections were blocked with 10% goat serum in PBS. The slides were then stained for CD8 (1:500, D4W2Z, Cell Signaling Technology), granzyme B (1:40, AF1865, R&D Systems), F4/80 (1:100, MCA497R, Bio-Rad), and OVA (1:500, AB1225, Millipore Sigma) for 16 hours at 4°C. Species-specific secondary antibodies conjugated to Cy5 or Cy7 fluorophores were used and incubated for one hour at room temperature in the dark. Sections were washed, counterstained with DAPI (100 ng/ml) and mounted using FluorSave (Calbiochem) mounting medium. Images were acquired on a Leica SP8 laser scanning confocal microscope using a 40x oil-immersion objective. Quantification of the fluorescent signals of the respective markers was performed using QuPath (69 (link)).
For immunofluorescence staining, muscle sections were de-paraffinized in xylene and rehydrated using a graded ethanol series culminating with PBS. Following antigen retrieval using a programmable pressure cooker with “target retrieval solution”, pH 6.0 (Dako), tissue sections were blocked with 10% goat serum in PBS. The slides were then stained for CD8 (1:500, D4W2Z, Cell Signaling Technology), granzyme B (1:40, AF1865, R&D Systems), F4/80 (1:100, MCA497R, Bio-Rad), and OVA (1:500, AB1225, Millipore Sigma) for 16 hours at 4°C. Species-specific secondary antibodies conjugated to Cy5 or Cy7 fluorophores were used and incubated for one hour at room temperature in the dark. Sections were washed, counterstained with DAPI (100 ng/ml) and mounted using FluorSave (Calbiochem) mounting medium. Images were acquired on a Leica SP8 laser scanning confocal microscope using a 40x oil-immersion objective. Quantification of the fluorescent signals of the respective markers was performed using QuPath (69 (link)).
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