C26.WT cells were cultured in a 24-well plate at a concentration of 100,000 cells/well. Then, the accumulation of ROS within cells was measured by labelling cells for 1 h at 37°C and 5% CO2 with 10 µM 5,6-chloromethyl-20,70-dichlorodihydrofluoresceindiacetate (CM-DCFDA; Molecular Probes; Thermo Fisher Scientific, Inc.; cat. no. C6827). The total cell number was determined by staining cells with 5 µl/ml Hoechst (Molecular Probes; Thermo Fisher Scientific, Inc.; cat. no. H1398) for 1 h at 37°C and 5% CO2. Finally, fluorescence was measured using a Fluoroskan Ascent plate fluorimeter (Thermo Labsystems, Santa Rosa, CA, USA), and data are expressed as a normalized percentage of CM-DCFDA/Hoechst fluorescence.
Hoechst
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Japan, Canada, Italy, Switzerland, Sweden, Belgium, France, China, Australia, New Zealand
Hoechst is a fluorescent dye used in laboratory applications for the detection and visualization of DNA. It binds to the minor groove of double-stranded DNA, emitting a blue fluorescent signal upon excitation. This property makes Hoechst a useful tool for various techniques, such as cell staining, flow cytometry, and fluorescence microscopy, where the quantification or localization of DNA is required.
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Lab products found in correlation
Triton x 100, by Merck Group (60 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (55 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (38 mentions)
Bovine serum albumin (bsa), by Merck Group (38 mentions)
Paraformaldehyde (pfa), by Merck Group (32 mentions)
Lsm 710 confocal microscope, by Zeiss (32 mentions)
Propidium iodide, by Thermo Fisher Scientific (31 mentions)
Axio observer z1, by Zeiss (29 mentions)
Lsm 700, by Zeiss (28 mentions)
Vectashield, by Vector Laboratories (26 mentions)
Calcein am, by Thermo Fisher Scientific (26 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (22 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (22 mentions)
Lsm 780 confocal microscope, by Zeiss (22 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (20 mentions)
Fluoromount g, by Southern Biotech (19 mentions)
Mitotracker green, by Thermo Fisher Scientific (18 mentions)
Lsm 780, by Zeiss (18 mentions)
Lsm 880 confocal microscope, by Zeiss (17 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (16 mentions)
1 591 protocols using hoechst
1
Measuring Cellular Oxidative Stress
2
Labeling and Tracking of Lipid Nanovesicles
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LNVs were labeled with PKH26 Red Fluorescent Cell Linker Kits (Merck KGaA, Darmstadt, Germany) following the datasheet information. Briefly, LNVs were incubated with PKH26 dye for 15 min at room temperature, washed twice in PBS, and resuspended in the growth medium. The labeled LNVs were incubated with HDFα cells for 4 h at 37°C with 5% CO2 and at 4°C. After incubation, the cells were fixed with PFA 4%, permeabilized with 0.1% TritonX-100, and stained with Actin Green (Molecular Probes, Life Technologies, Carlsbad, CA, USA) and Hoechst (Molecular Probes, Life Technologies, Carlsbad, CA, USA).
HDFα were treated with LNVs (10 and 25 μg/ml) for 24h, then the cells were fixed with PFA 4%, permeabilized with 0.1% TritonX-100, incubated with anti-AhR antibody (Novus Biologicals, Milano, Italy) or anti-Nrf2 antibody (Novus Biologicals, Milano, Italy) for 1h, then washed and incubated with Goat anti-Rabbit IgG Secondary Antibody, DyLight 594 or 488 (Invitrogen) for 1h. Actin was stained using Actin Green (Molecular Probes, Life Technologies, Carlsbad, CA, USA), and nuclei were stained with Hoechst (Molecular Probes, Life Technologies, Carlsbad, CA, USA).
The samples were analyzed by confocal microscopy (Nikon A1, Amsterdam, Netherlands).
HDFα were treated with LNVs (10 and 25 μg/ml) for 24h, then the cells were fixed with PFA 4%, permeabilized with 0.1% TritonX-100, incubated with anti-AhR antibody (Novus Biologicals, Milano, Italy) or anti-Nrf2 antibody (Novus Biologicals, Milano, Italy) for 1h, then washed and incubated with Goat anti-Rabbit IgG Secondary Antibody, DyLight 594 or 488 (Invitrogen) for 1h. Actin was stained using Actin Green (Molecular Probes, Life Technologies, Carlsbad, CA, USA), and nuclei were stained with Hoechst (Molecular Probes, Life Technologies, Carlsbad, CA, USA).
The samples were analyzed by confocal microscopy (Nikon A1, Amsterdam, Netherlands).
3
Mitochondrial Stress Assay in Myotubes
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Myotubes were treated with 4-HNE overnight. Stained with 200 nM MitoTracker Green FM (Molecular Probes) for 45 min and 2 μg/mL Hoechst (33,342, Molecular Probes) for 10 min at 37 °C. Fluorescence intensity of MitoTracker was measured using TECAN Infinite M200 PRO plate reader (Ex 490 nm/ Em 516 nm). Myotubes were imaged using ArrayScan XTI (Thermo) with 386-23 nm filter for Hoechst and 485-20 nm filter for MitoTracker.
4
Immunostaining of the Placozoan Trichoplax
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T. adhaerens were left to adhere at the bottom of 35-mm plastic Petri dish filled with ASW, swiftly replaced by 4% paraformaldehyde (PFA) with Dextran in ASW for fixation (1-2 hr, RT). After brief washes in ASW followed by 0.1 M phosphate buffer pH7.4 (PB) and a 1 hr-blocking step in 3% normal goat serum (NGS) + 0.2% Triton X-100 in PB, they were incubated for 2 hr in primary antibody (antipeptide: 1 mg/mL; anti-FMRFamide (1:300; Enzo Life Sciences #BML-FA1155 or Phoenix Pharmaceuticals #H-047-29) in blocking solution. After thorough washes in PB, a 1-hr incubation in the corresponding secondary antibody coupled to a fluorophore (1:600, GAR-Alexa Fluor, Molecular Probes) and further washes in PB, they were incubated for 2 min in Hoechst (1:10,000; Molecular Probes), rinsed and mounted with Prolong-Gold (LifeTech) on glass slides. For co-labeling with antibodies raised in the same species, T. adhaerens were instead incubated 45 min in a mix of antibodies coupled to Alexa fluorophores using the Zenon labeling kit (LifeTechnologies) following the manufacturer's instructions, washed in PB, post-fixed 15 min in 4% PFA, washed again and incubated in Hoechst before being mounted on a slide. For blocking experiments, we pre-incubated the antibodies in three times excess of the respective peptides for 2 h before immunostainings.
5
Mitochondrial and Lysosomal Staining Imaging
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For the mitochondrial staining, after washing once with PBS, 5 mM Mitotracker (Thermo Fisher Scientific) and 200 mM Hoechst (Thermo Fisher Scientific) were added to the cells. For lysosome staining, 5 μM LysoMitotracker (Thermo Fisher Scientific) and 200 mM Hoechst (Thermo Fisher Scientific) were added to the cells. After incubation for 1 hour, a z-stack fluorescence image was obtained using a fluorescence microscope. MitoSOX Red (Thermo Fisher Scientific) was applied to measure the superoxide anion levels. The kidney organoids were treated with Hoechst 33342 (200 mM) for 1 hour and MitoSOX Red (5 μM) for 15 minutes at 37°C in the dark and washed with PBS. A z-stack fluorescence image was obtained using a fluorescence microscope.
6
Nrp1 Modulates Myoblast-Schwann Cell Fusion
7
Immunostaining of Cultured Cells
8
Visualizing VSV-GFP Infection and Cellular Uptake
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The VSV-GFP is a recombinant virus expressing the green fluorescent protein. The Cy3 fluorescein was modified on the RVG to provide a red fluorescent signal. In the cellular model, the virus-infected N2a cells (MOI of 1) were incubated with 100 μL of T-705@MSN-RVG (2 mg/mL) at 37 °C for 1h. Then, cells were washed three times and stained by a Hoechst (Thermo Fisher, Waltham, MA, USA) at 37 °C for 5 min. Subsequently, the cells were visualized by a TS100 fluorescence microscope (Nikon, Tokyo, Japan).
In the mouse infection model, co-localization was determined in the mouse brain. Briefly, six-week-old female Balb/c mice were infected with VSV-GFP at 200 FFU or DMEM by intracranial (i.c.) route. Then, the mice were inoculated with 25 μL of T-705@MSN-RVG (1 mg/mL) by i.v. injection. The mouse brains were collected at 2 dpi and fixed in 4% paraformaldehyde for 48 h, followed by being dehydrated in 30% sucrose solution and embedded in SAKURA Tissue-Tek® O.C.T compound (SAKURA, Torrance, CA, USA) for rapid freezing and slicing [35 (link)]. The slices were stained by Hoechst (Thermo Fisher, Waltham, MA, USA) at 37 °C for 5 min and then visualized by a TS100 fluorescence microscope (Nikon, Tokyo, Japan).
In the mouse infection model, co-localization was determined in the mouse brain. Briefly, six-week-old female Balb/c mice were infected with VSV-GFP at 200 FFU or DMEM by intracranial (i.c.) route. Then, the mice were inoculated with 25 μL of T-705@MSN-RVG (1 mg/mL) by i.v. injection. The mouse brains were collected at 2 dpi and fixed in 4% paraformaldehyde for 48 h, followed by being dehydrated in 30% sucrose solution and embedded in SAKURA Tissue-Tek® O.C.T compound (SAKURA, Torrance, CA, USA) for rapid freezing and slicing [35 (link)]. The slices were stained by Hoechst (Thermo Fisher, Waltham, MA, USA) at 37 °C for 5 min and then visualized by a TS100 fluorescence microscope (Nikon, Tokyo, Japan).
9
Mitochondrial Content and ROS Quantification
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The mitochondrial DNA (mtDNA), an index of mitochondrial content (mtDNA copy number), was carried out in brown adipose tissue as previously validated and described [31 (link)]. Briefly, DNA was extracted using QIAzol® (Qiagen) protocol. Real-time PCR was performed to amplify the mitochondrial gene 16S which is exclusive of mtDNA, and a nuclear gene (mtDNA/nDNA ratio). In cultured brown adipocytes, Mitotracker Green (Thermo Fisher Scientific, Waltham, MA, USA) was used at 200 nM for 90 min, meanwhile Hoechst (Thermo Fisher Scientific, Waltham, MA, USA) was used at 2 μg/μl for 10 min followed by wash-out before imaging (Zeiss LSM 710 confocal microscope). Mitotracker Green was excited by a 488 nm laser, while 361 nm laser was used for detection of Hoechst. Imaging was performed with a 40× objective. The dye signal was quantified with the Image J analysis software and normalized to the number of cells (Hoechst dye). The ROS assay was performed as previously described [32 (link)]. Values were expressed as the ratio between the fluorescence of the oxidized CM-H2DCFDA (Thermofisher) and Hoechst, after subtraction of the signal of not-stained cells.
10
Visualization of Hepatic Canaliculi in Organoids
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Staining with CDFDA was performed to visualize active bile canaliculi in the hepatic organoids co-cultured with microvessels. Briefly, a stock solution containing 5 mM 5-(and-6)-carboxy-2′,7′-dichloro-fluorescein diacetate (CDFDA) (Sigma-Aldrich, #2188) was prepared in 100% Dimethyl Sulfoxide (DMSO) (Sigma, #D8418), aliquoted, and stored at -20 °C. For the assay, culturing media were aspirated in all graft chambers and perfusion in- and outlets and 50 µL of Hep-Medium containing 5 µM CDFDA (1:1000), and Hoechst (Thermo Fisher Scientific, #H3570, 1:2000 in PBS) was added to each well. The OrganoPlate Graft was incubated for 30 min in the incubator (37 °C, 5%, CO2) on an interval rocker switching between a + 14° and − 14° inclination every 8 min (OrganoFlow S, Mimetas). As negative control, Hep-Medium media containing 0.1% DMSO and Hoechst (Thermo Fisher Scientific, #H3570, 1:2000 in PBS) was prepared. At the end of the incubation, chips were washed 6 times with PBS and imaged Micro XLS-C High Content Imaging Systems (Molecular Devices, US).
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