Nile red–labeled beads (20 nm diameter; Invitrogen, MA, US) were diluted with distilled water (1:2,500–1:5,000, v/v). The bead solutions were added dropwise to high-tolerance coverslips (0.17 ± 0.005 mm thickness; Matsunami, Osaka, Japan), followed by drying and mounting using a ProLong diamond reagent (Invitrogen). Fluorescent images of the beads were obtained at a pixel size and dwell time of 28 nm × 28 nm and 40 μs, respectively.
Prolong diamond reagent
Manufactured by Thermo Fisher Scientific
Sourced in Japan, United States
Prolong Diamond reagent is a mounting medium designed for preserving fluorescence in microscopy samples. It is formulated to reduce photobleaching and maintain the brightness of fluorescent labels over time.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Tcs sp5 laser scanning microscope, by Leica (1 mentions)
Il 1β, by Cell Signaling Technology (1 mentions)
Nlrp3, by Adipogen (1 mentions)
Caspase 1, by Adipogen (1 mentions)
Nile red, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Fv1000 d confocal microscope, by Olympus (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Alexa fluor 488 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti phospho histone h3 s10, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
Fv10 asw software, by Olympus (1 mentions)
Ab254530, by Abcam (1 mentions)
Lsm710 laser scanning confocal microscopy, by Zeiss (1 mentions)
Pro spc, by Abcam (1 mentions)
Seaplaque agarose, by Lonza (1 mentions)
7 protocols using prolong diamond reagent
1
Nile Red Bead Fluorescence Imaging
2
Immunohistochemical Analysis of NLRP3, Caspase-1, and IL-1β in Liver Tissue
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The livers tissues were post-fixed in fresh 4% paraformaldehyde overnight at 4 °C and cryoprotected with 30% sucrose solution for 24 h. Histological sections (thickness 16 μm) of paraformaldehyde-fixed liver samples were treated with a serum-free protein block reagent (Agilent Technologies, Santa Clara, CA, USA) and incubated with primary antibodies against NLRP3 (AdipoGen Life Sciences, San Diego, CA, USA), caspase-1 (AdipoGen Life Sciences, San Diego, CA, USA) and IL-1β (Cell Signaling Technology, Danvers, Massachusetts, USA) overnight at 4 °C in a humidified chamber. Slides were washed in phosphate buffered saline (PBS), incubated with secondary anti-mouse antibodies for 1 h, mounted with ProLong Diamond reagent (Invitrogen, Carlsbad, CA, USA) for visualization by the Leica TCS SP5 laser scanning microscope (Leica Microsystems, Wetzlar, Germany). Negative controls were run by omitting the primary antibody step.
3
Fluorescent Bead Imaging and Tubulin Staining
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Three types of fluorescently-labeled beads (Nile Red, ~1 μm diameter; Nile Red, ~20 nm diameter; and Red, ~100 nm diameter; Thermo Fisher Scientific) were diluted in water (1:3000, v/v), applied dropwise to glass coverslips, and allowed to dry. The coverslips were then mounted using a mounting medium.
Next, HeLa cells were cultured on glass coverslips in Dulbecco’s modified Eagle’s medium (DMEM; Wako Pure Chemical, Co.), supplemented with 10% fetal bovine serum and penicillin/streptomycin (Thermo Fisher Scientific), at 37°C in a humidified atmosphere containing 5% CO2. The cells were then stained with an anti-α-tubulin antibody (clone DM1A; Cell Signaling Technology) and an ATTO 532-conjugated anti-mouse IgG antibody (Rockland Immunochemicals, Inc.), and mounted using ProLong diamond reagent (Thermo Fisher Scientific).
Next, HeLa cells were cultured on glass coverslips in Dulbecco’s modified Eagle’s medium (DMEM; Wako Pure Chemical, Co.), supplemented with 10% fetal bovine serum and penicillin/streptomycin (Thermo Fisher Scientific), at 37°C in a humidified atmosphere containing 5% CO2. The cells were then stained with an anti-α-tubulin antibody (clone DM1A; Cell Signaling Technology) and an ATTO 532-conjugated anti-mouse IgG antibody (Rockland Immunochemicals, Inc.), and mounted using ProLong diamond reagent (Thermo Fisher Scientific).
4
Multiparametric Analysis of DNA Damage and Mitosis
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The cells were fixed with ice‐cold methanol for 15 min for staining γ‐H2AX and with 4% paraformaldehyde for staining mitotic cells and YAP/TAZ, respectively. The fixed cells were permeabilised with 0.3% Triton X‐100 in PBS and blocked with 3% fetal bovine serum in PBS for 30 min. They were incubated with 1/1000 rabbit anti‐γ‐H2AX (Genetex, Irvine, CA, USA; GTX127340), 1/2000 mouse anti‐α‐tubulin (Sigma‐Aldrich; B‐5‐1‐2), 1/500 anti‐phospho‐Histone H3 S10 (Cell Signaling Technology; #9701) or 1/300 rabbit anti‐YAP/TAZ antibody (Cell Signaling Technology; #8418) at 4 °C overnight and then washed with PBS three times. Next, the cells were incubated with 1/500 anti‐rabbit IgG‐Alexa Fluor 488, 1/500 anti‐mouse IgG‐Alexa Fluor 488, anti‐rabbit IgG‐Alexa Fluor 594 or phalloidin‐Alexa Fluor 594 (Thermo Fisher) for 1 h at room temperature and again washed with PBS three times. Then, the cells were mounted in Prolong Diamond reagent containing 10 μg·mL−1 of Hoechst 33342 (Thermo Fisher Scientific). Images were obtained with an FV1000‐D confocal microscope using fv10‐asw software (Olympus, Tokyo, Japan). To capture mitotic cells, ~ 40 images were collected with a z‐optical spacing of 0.2 μm with a 100× numerical aperture 1.4 objective lens. For other cells, single sections were collected with a 40x objective lens.
5
Multiplexed IHC of CRC Organoids
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Formalin‐fixed paraffin‐embedded CRC organoids were cut at a thickness of 5 µm onto slides. The slides were then baked at 65 °C for 2 h and immersed in xylene for 15 min in duplicate. The slides were then immersed in 100%, 95%, and 75% ethanol followed by water for 3 min. Then, antigen retrieval was performed with sodium citrate (pH 6.0). After washing with water, the slides underwent multiple rounds of multiplex fluorescent immunohistochemistry with the Opal Multiplex Fluorescent Immunohistochemistry kit (Perkin Elmer). The following primary antibodies and subsequent Opal TSA combinations were used: anti‐CD44 (1:250, abcam, ab254530) and Opal 520, anti‐ki67 (1:250, abcam, ab92742) and Opal 570, anti‐CEA (1:400, bioss, bs‐0719R) and Opal 620, anti‐EP‐CAM (1:250, Santa Cruz, sc‐53532) and Opal 690, and DAPI. The slides were mounted on coverslips with Prolong Diamond reagent (ThermoFisher).
6
Immunohistochemical analysis of lung tissue
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The lung prepared as described above was instilled with 1 ml 4% paraformaldehyde in PBS(+) and fixed with 4% paraformaldehyde for 2 days at room temperature. The lung was embedded in 5% Seaplaque agarose (Lonza) or paraffin and then sectioned to 100 μm or 6 μm thickness, respectively, with a microslicer (DOSAKA EM). Paraffin sections were deparaffinized with xylene and ethanol and then rehydrated. The obtained sections were permeabilized with 0.5% TritonX-100 in PBS(+) for 1 h, stained with specific antibodies against HA (C102), FITC, ABCA3 (3C9), or Pro-SP-C (Abcam), followed by Alexa488-labeled or Alexa546-labeled secondary antibodies, and then mounted with ProLong Diamond reagent (Thermo Fisher Scientific). All images were analyzed using LSM710 laser scanning confocal microscopy (Zeiss).
7
3D DNA FISH on Fibro-Adipogenic Progenitors
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Fluorescent in situ hybridization (FISH) was carried out on cells that adhered to glass coverslips coated with ECM gel (Sigma-Aldrich) using nick-translated BAC DNA (BAC RP23-471-MyoD Locus) that incorporate Cy 3–deoxyuridine triphosphate (Enzo Life Sciences). Three-dimensional (3D) DNA FISH on FAPs was performed with some modifications of already described procedures (69 (link)). Briefly, cells were fixed in 4% PFA (20 min at 4°C) and preprocessed by freeze-thawing permeabilization to ensure the preservation of nuclear structures. After denaturation, FISH probes were hybridized overnight at 37°C, and samples were washed, DAPI-stained, and mounted in ProLong Diamond reagent (Thermo Fisher Scientific). Samples were imaged on inverted microscope (Olympus IX73) equipped with a confocal imager (Crest X-Light) spinning disk, a CoolSNAP MYO charge-coupled device camera (Photometrics), and a Lumencor Spectra X light-emitting diode illumination. Images were acquired with 60× NA 1.35 oil objective (UPlanSAPO) and MetaMorph (Molecular Devices) using 300 ms (Cy3), 300 ms (fluorescein), or 100 ms (DAPI) as exposure time. Confocal images were taken with a 0.2-μm-step Z-stacks. 3D reconstructions and 3D distance between the center of mass of the DNA FISH spots and the inner surface of the lamina were performed by using Huygens Professional software.
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