Following treatment, growth medium containing 100 nM MitoTracker probe® (Molecular Probes, Eugene, OR, USA) was added to the culture, which was incubated for 30 min at 37°C. The cells were washed with PBS and then fixed in 4% formaldehyde in PBS for 15 min at 37°C. After washing the cells, they were permeabilized with 0.1% Triton X-100 in PBS for 15 min at 4°C. To prevent nonspecific binding, the cells were incubated in blocking buffer (1% BSA in PBST, pH 7.4) for 30 min at room temperature. Anti-cytochrome c antibody (0.5 μg/mL; 2 mL) (Abcam, Cambridge, MA, USA) was added and incubated for 1 h at room temperature. The coverslip was washed and then incubated in 2 mL Hoechst 33258 stain (1 μg/mL in PBS) (Sigma-Aldrich, St. Louis, MO, USA) for 20 minutes. The coverslip was washed again before being mounted with a drop of mounting medium (90% glycerol in PBS) and sealed with nail polish and stored in the dark at 4°C. The slide was examined on the Axioplan 2 fluorescence microscope (Zeiss, Jena, Germany) at 630x magnification and micrographs of the cells were taken using AxioVision 4.8 software. The excitation and emission wavelengths (nm) of the fluorescent dyes were as follows: Alexa-488, excitation: 499 and emission: 519; MitoTracker orange probe, excitation: 554 and emission: 576; and Hoechst 33258, excitation: 343 and emission: 483.
Hoechst 33258 stain
Manufactured by Merck Group
Sourced in United States, Germany
Hoechst 33258 is a fluorescent dye commonly used in molecular biology and cell biology applications. It binds selectively to adenine-thymine (A-T) rich regions in DNA, resulting in a blue fluorescence when exposed to ultraviolet (UV) light. This property makes Hoechst 33258 a useful tool for visualizing and quantifying DNA in various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 labeled goat anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Anti h3k9me3, by Merck Group (2 mentions)
Optical bottom tissue culture plates, by Thermo Fisher Scientific (2 mentions)
Anti h3k36me2 ab9049, by Abcam (2 mentions)
Arrayscan vti high content screen reader, by Thermo Fisher Scientific (2 mentions)
Mitotracker probe, by Thermo Fisher Scientific (1 mentions)
Anti cytochrome c antibody, by Abcam (1 mentions)
Axioplan 2 fluorescence microscope, by Zeiss (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid bca protein assay kit, by Beyotime (1 mentions)
Fluorescein diacetate fda, by Merck Group (1 mentions)
Irdye 680lt goat anti mouse, by LI COR (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
11 protocols using hoechst 33258 stain
1
Visualizing Mitochondrial Cytochrome c by Immunofluorescence
2
Apoptosis Assay in MCF-7 Cells
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Nuclear chromatin condensation is a critical feature characterizing apoptosis. MCF-7 at a concentration of 1 × 105 cells/mL per well was added to 24-well plate, and it was incubated for 24 h. The old medium was replaced and EHX was added at a concentration of 7.5, 15, and 30 μg/mL or absolute ethanol for the control sample. The plate was reincubated for another 24 h. The medium was discarded, cells were rinsed by PBS, and para-formaldehyde 4% (w/v) was added for cell fixation. After 20 min, Hoechst 33258 stain (10 μg/mL; Sigma) was added to each well (300 μL/well), and the plate was incubated for a further 20 min. Microimages were snapped under 20x magnification using a fluorescence microscope (EVOS fl, USA) [23 (link)].
3
Screening Histone Methylation Modulators
4
Apoptosis and Oxidative Stress Assays
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Rhodamine 123 (Rh123), cadmium acetate (CdAc2), and Hoechst 33258 stain were purchased from Sigma Chemical (USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco Laboratories (USA). Fluorescein isothiocyanate (FITC)-annexin V apoptosis detection kits were purchased from BD Biosciences Pharmingen (USA). Lactate dehydrogenase (LDH) and SOD assay kits were acquired from Jiancheng Bioengineering Institute (China). GR activity and caspase-3/CPP32 fluorometric assay kits were both obtained from BioVision Research Products (USA). Cell Counting Kit-8 (CCK8), ALP staining kits and bicinchoninic acid (BCA) protein assay kits were provided by the Beyotime Institute of Biotechnology (China). Anti-rat c-jun N-terminal kinase (JNK), P-JNK, extracellular signal-regulated kinase (ERK), P-ERK, p38, P-p38, β-actin, cleaved poly (ADP-ribose) polymerase (PARP), cleaved caspase-9, cleaved caspase-3, Bax, Bcl-2, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibodies were obtained from Cell Signaling Technology (USA).
5
Antibody and Reagent Sources for Oxidative Stress Research
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Antibodies and reagents were obtained as follows: anti-heme oxygenase-1 (HO-1) polyclonal rabbit IgG (OSA-150, Assay Design, Ann Arbor, MI), anti-NADPH quinone oxidoreductase1 (NQO1) polyclonal rabbit IgG (2618-1, Epitomics, Cambridge, MA), anti-HSP70 monoclonal mouse IgG (200-301-A27, Rockland, Pottstown, PA), IRDye 800CW goat anti-rabbit (green fluorescent; LI-COR, Lincolon, NE, catalogue number 926-32211), and IRDye 680LT goat anti-mouse (red fluorescent; LI-COR, Lincolon, NE, catalogue number 926–68020). Other reagents including dimethylsulfoxide (DMSO), sodium glutamate, fluorescein diacetate (FDA), hydrogen peroxide (HP), tunicamycin (TM), and Hoechst 33 258 stain were obtained from Sigma (St Louis, MO). The chemical synthesis of D1 has been described previously (Satoh et al., 2011 (link)). Synthesis of analogues D1 and D3 are described later.
6
Immunofluorescent Analysis of S100P
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Cell monolayers cultured in 96-well dishes or on coverslips were fixed with 4% paraformaldehyde. After permeabilization with 0.1% Triton X-100 for 45 min, cells were incubated with 1:50 mouse anti-human S100P monoclonal antibody (R&D Systems, Minneapolis, MN, USA) as the primary antibody, and then incubated with 1:500 Cy3-labeled goat anti-mouse IgG (Invitrogen) as the secondary antibody. Alternatively, the cells were stained with 5 μg/ml FITC-phalloidin (Sigma) for F-actin staining. Cell nuclei were counterstained with Hoechst33258 stain (Sigma) at 1 mg/ml. The cells were then observed under a fluorescence microscope (Axiovert 200, Zeiss, Jena, Germany).
7
Immunohistochemical Staining of Cx43 in Frozen Heart Sections
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Unfixed frozen hearts for immunohistochemical stainings were embedded in TissueTek OCT medium (Sakura Finetek, Germany) and processed to 10 μm cryosections. After fixing with 4% PFA in PBS, sections were washed three times with 0.1% Tween in TBS and blocked with 5% BSA, 0.1% Tween in TBS for one hour at room temperature. Sections were then incubated with rabbit anti-Cx43 serum (1:1000) diluted in blocking solution at 4°C overnight. On the next day, the sections were washed three times with 0.1% Tween in TBS and incubated with secondary donkey anti-rabbit antibody conjugated with Alexa 488 (1:1000, Molecular Probes, Invitrogen, USA) diluted in blocking solution for one hour at room temperature. After washing the sections twice with 0.1% Tween in TBS, nuclei were stained by incubating sections in 0.1% Tween in TBS with 0.5 mg/ml bisbenzimide (1:1000, Hoechst 33258 stain; Sigma, Germany) for 10 minutes at room temperature. Sections were mounted with Dako Glycergel mounting medium (Dako North America, USA) and viewed with a confocal Laser Scanning Microscope (LSM 710 DuoScan, Zeiss, Germany).
8
Assessing Plasmodium Invasion Inhibition
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The effect of mouse sera and rPfRH5 on the parasite invasion of human red blood cells (RBCs) was assessed in three experiments using an invasion inhibition assay. Synchronized trophozoites of the P. falciparum CY and 3D7 strains were purified by centrifugation at 1,500 × g and 4°C for 15 min on Percoll gradients (16 (link)). To obtain a final hematocrit of 2% and parasitemia of 0.2%, ~7.2×105 schizonts in 180 ml complete medium with 10% human serum were mixed with 4×107 human RBCs. Each well received 180 μl of the culture and 20 μl heat-inactivated (56°C, 30 min) mouse anti-PfRH5 sera (or rPfRH5 protein). The medium was changed every 24 h. After incubation for 72 h, the number of parasites was determined; the parasites were collected by centrifugation (3 min at 1,000 × g) and the supernatant was removed by aspiration (17 (link)). Hoechst 33258 stain (no. 861405; Sigma-Aldrich) in PBS (0.1 ml) was added to each well to cover the complete surface of the well and left for 10 min at 37°C. The stain was removed by centrifugation and the RBCs were washed twice with PBS. Fluorescence intensity was read at 450 nm after 10 min using a GENios Pro Reader (Tecan, Männedorf, Switzerland). The percent inhibition was calculated using the fluorescence intensity data in the pre-immunization serum controls as 100% invasion.
9
Quantifying Mitotic Activity via PHH3 Immunostaining
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Increased pHH3 levels are associated with increased mitotic activity and can be used as an indicator of malignancy [23 (link)]. Tissue sections were incubated in 3% hydrogen peroxide for 30 min to block endogenous peroxidases, rinsed once in deionized H2O, and followed with three times in PBS. Sections were then incubated with 3% normal goat serum in PBS for 1 hour to block nonspecific binding proteins. Retrieved sections were placed overnight at 4°C in a moist chamber with rabbit polyclonal anti-PHH3 at a dilution 1 : 500 (Millipore, Billerica, MA, USA). After overnight incubation, slides were rinsed twice in PBS (10 min each) and incubated with goat anti-rabbit fluorescence antibodies at 1 : 500 dilutions (F2765, Invitrogen Molecular Probes, Carlsbad, CA) for 1 hour. Slides were rinsed twice in PBS (10 min each), and nuclei were then distinguished by counterstaining with Hoechst 33258 stain (Sigma-Aldrich, Darmstadt, Germany).
10
High-Throughput Screening of Histone Methylation
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HeLa and MiaPaCa2 cells were seeded in 96-well optical bottom tissue culture plates (165305; Thermo Scientific, Waltham, MA) in Dulbecco’s modified Eagle’s medium (DMEM). Six hours later, 1 µL of a serial dilution of compounds was added in each well (50 µM to 25 nM). Similar dilutions of methylstat acid14 (link) and DMSO were used as positive and negative controls, respectively. After incubation for 48 h, cells were fixed in 4% paraformaldehyde in Dulbecco’s phosphate buffered saline (D-PBS), permeabilized, blocked with bovine serum albumin (BSA), and then treated with methylation-specific antibodies, rabbit polyclonal anti-H3K36me2 (ab9049; Abcam, Cambridge, MA), or rabbit polyclonal anti-H3K9me3 (07-442; Millipore, Billerica, MA), respectively, followed by Alexa Fluor 488–labeled goat–anti-rabbit secondary antibody (Invitrogen, Carlsbad, CA) and Hoechst 33258 stain (Sigma, St. Louis, MO). Images were captured and analyzed using the ArrayScan VTI High Content Screen (HCS) Reader (Thermo Scientific).
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