Cells were fixed with 4% buffered formalin at room temperature for 10 min. Cell permeabilisation was performed using 0.15% Triton®-X100 in PBS. Horse serum (2% v/v) was used to inhibit non-specific binding. Cells were stained with β-Catenin XP® Rabbit mAb (cat. no. 8480, Cell Signaling, Danvers, MA, USA) at a 1:100 dilution at 4 °C overnight. Cells were incubated with biotinylated anti-rabbit IgG antibodies (cat. no. 2172707, Sigma-Aldrich, CA, USA) at a dilution of 1:2000 for 40 min. The targeted protein expression was visualised by staining with Strep-FITC (Sigma-Aldrich, CA, USA) at a dilution of 1:500. Nuclei were counterstained with 0.1 μg/mL 4′,6-diamidino-2-phenylindole (TOCRIS Bioscience, Minneapolis, MN, USA). Protein expression and localisation were performed using a fluorescent microscope with an ApoTome system (Carl Zeiss, Oberkochen, Germany).
4 6 diamidino 2 phenylindole (dapi)
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) is a fluorescent dye used for nucleic acid staining. It binds strongly to DNA and can be used to visualize cellular nuclei in a variety of applications, including fluorescence microscopy and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Strep fitc, by Merck Group (3 mentions)
Apotome system, by Zeiss (2 mentions)
Edc4 ge 1 rabbit antibody, by Cell Signaling Technology (2 mentions)
Alexa fluor 488 conjugate anti rabbit igg h l f ab 2 fragment, by Cell Signaling Technology (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Lsrfortessa, by BD (1 mentions)
Facsaria fusion, by BD (1 mentions)
Brilliant stain buffer, by BD (1 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Fv3000 confocal laser scanning microscope, by Olympus (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Ccd camera, by Hamamatsu Photonics (1 mentions)
Tunel assay kit, by Abcam (1 mentions)
Mouse anti collagen 1, by Merck Group (1 mentions)
Dako anti fading reagent, by Agilent Technologies (1 mentions)
Dylight 550, by Thermo Fisher Scientific (1 mentions)
Dylight 650, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Red blood cell lysis buffer, by Merck Group (1 mentions)
18 protocols using 4 6 diamidino 2 phenylindole (dapi)
1
Immunofluorescence Staining of β-Catenin
2
Immunofluorescent Localization of β-Catenin
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Cells were fixed with 4% buffered formalin at room temperature for 10 min and permeabilized using 0.15% Triton-X100 in PBS. Horse serum (2% v/v) was used to inhibit a non-specific binding. Cells were stained with β-Catenin XP Rabbit mAb (cat. no. 8480, Cell Signaling, USA) at a 1:100 dilution at 4 °C overnight. Cells were incubated with biotinylated anti-rabbit IgG antibodies (cat. no. 2172707, Sigma-Aldrich, USA) at dilution 1:2000 for 40 min. The targeted protein expression was visualized by stained with Strep-FITC (Sigma-Aldrich, USA) at dilution 1:500. Nuclei were counterstained with 0.1 μg/mL 4′,6-diamidino-2-phenylindole (TOCRIS bioscience, USA). Protein expression and localization was detected using a fluorescent microscope with ApoTome system (Carl Zeiss, Germany).
3
Cell Dissociation and Antibody Labeling Protocol
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Cells were dissociated with Accutase, washed with 2% FBS in PBS (wash buffer) and filtered through a 50 µm sterile strainer (Sysmex). Antibody labelling was performed by incubating cells in a Brilliant Stain Buffer (BD Biosciences) with antibodies for 30 min at 4°C in the dark. This was followed by a wash in wash buffer, cell pelleting at 300 g for 3 min and re-suspending the cells in 300 µl of the wash buffer. To identify live and dead cells, 0.1 µg/ml DAPI (Tocris) or Fixable Viability Dye eFluor 780 (eBioscience) was used. Antibody details are listed in Table S7 . Flow cytometry analysis was performed on a BD LSR-Fortessa at the Babraham Institute Flow Core. Cell-sorting experiments were performed on a BD Influx or a BD FACSAria Fusion. Data processing and downstream analysis were performed using FlowJo V10.1.
4
Quantifying Microglia and Astrocyte Distribution in Brain Tissue
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The lower left hemispheres of the brains were incubated with 4% paraformaldehyde for 1 week at room temperature, then moved into a 30% sucrose solution and kept at 4°C until further analysis. Brain slices of 20‐μm thickness were cut by cryosectioning and incubated with 3% peroxide for 1 hour, followed by an overnight incubation with a solution of BSA and primary antibody against Iba‐1 (Abcam, Cambridge, UK), glial fibrillary acidic protein (Abcam, Cambridge, UK), and DAPI (TOCRIS, Bristol, UK). PBS was used to wash the brain slices clear of unbound primary antibody, then AlexaFluor 488 anti‐goat Iba1 and AlexaFluor 647 anti‐rabbit glial fibrillary acidic protein conjugated secondary antibodies were incubated with the samples for 1 hour. A FV3000 confocal laser scanning microscope (Olympus Corp., Tokyo, Japan) was used to acquire images of the brain slices every 0.5 μm in the z‐plane. The images were analyzed using Imaris Image Analysis Software (Bitplane, Belfast, Northern Ireland).
5
Immunofluorescence Imaging of HMGB1 and ASC
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BMDMs cultured on 18 mm glass coverslips in 12-well plates (150,000 cells per well) and treated as indicated in the figure legends, washed with PBS and fixed in 3% paraformaldehyde at RT for 15 min. The cells were washed three times in PBS, permeabilized with 0.1% TX-100, then incubated in 2%BSA/PBS for 1 h. Primary antibodies were then added for 1 h at RT or overnight at 4 °C. The cells were washed three times with PBS and Cy3 or Alexa488-conjugated secondary antibodies was added for 1 h at room temperature (1:200). The cells were then washed with PBS, incubated for 5 min in 0.5 μM DAPI (Tocris #5748), washed with PBS. Cells were imaged by spinning disk confocal microscopy (Quorum) on a Zeiss Axiovert 200M microscope with a ×63 (or ×25) objectives and an additional ×1.5 magnifying lens. Images were acquired by a CCD camera (Hamamatsu Photonics) driven by the Volocity software. Primary Antibodies used: rabbit anti-human HMGB1 (Abcam ab79823; 1:100) and mouse anti-ASC (Biolegend, Cat: 653902; 1:100).
6
Histological Analysis of Apoptosis in Liver
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Liver tissues were embedded in paraffin, and serial 5-μm-thick sections were placed onto slides. After dewaxing and rehydration, the sections were stained with H&E according to routine protocols. For the TdT-mediated TUNEL assay, the sections were deparaffinized, and apoptotic cells were detected using the in situ BrdU-Red DNA fragmentation (TUNEL) assay kit (Abcam，cat. #ab66108) and counterstained with DAPI (Tocris, cat. #5748).
7
Immunofluorescent Assay for Collagen I
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Cells were fixed with 4% buffered formalin for 10 min. Horse serum (2% v/v) was used to inhibit a non-specific binding. Cells were stained with mouse anti-collagen I (C2456, Sigma) at 4 °C overnight and subsequently incubated with biotinylated anti-mouse antibodies (Invitrogen) for 40 min. The targeted protein expression was visualized by stained with Strep-FITC (Sigma). DAPI (TOCRIS bioscience) was employed for nuclei staining.
8
Quantification of Islet Apoptosis
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Pancreas sections obtained as described in Section 2.3 were used for the determination of islet apoptosis, assessing DNA fragmentation by the Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) method, following the manufacturer’s instructions (Fluorometric DeadEndTM TUNEL System, Promega). In the same procedure, pancreas sections were stained with DAPI (Tocris, Bioscience, MN, USA), anti-mouse insulin antibody (1/200, Polyclonal Guinea Pig Anti-Insulin A564, Dako Agilent, Santa Clara, CA, USA) and Alexa Fluor 633 goat anti-guinea pig (Thermo Fisher Scientific) antibodies in order to identify and quantify the TUNEL-positive cells inside the islets using the ImageJ software. Samples were treated with Dako anti-fading reagent (Dako, DAgilent, Santa Clara, CA, USA) and stored at 4°C until their analysis by immunofluorescence microscopy.
9
Immunofluorescent Labeling of Cellular Markers
10
Immunofluorescence Staining of G3BP1
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