Thymoquinone (TQ) was product of MCE (CAS: 490-91-5). Hoechst 33342 (B2261) was bought from Sigma-Aldrich. Cell counting kit‐8 (CCK‐8) was product of Selleck Chemicals (US). N-Acetyl-L-cysteine (NAC) and Z‐VAD‐FMK (ZVF) were obtained from Beyotime Biotechnology (Shanghai, China). The Apoptosis Detection Kit #556547 was purchased from BD (San Jose, CA). Antibodies against Bax (ab32503), Bcl‐2 (ab182858), LC3B (ab192890), Beclin-1 (ab207612), ATG7 (ab133528), MMP2 (ab92536), MMP9 (ab76003) and PD-L1 (ab213524) were bought from Abcam (San Francisco, CA). Antibody against vimentin (V6389) was purchased from Sigma-Aldrich (US). Antibodies against β‐actin (20536‐1‐AP) and Bcl-xl (10783-1-AP) were obtained from Proteintech (Chicago, IL). Antibodies against cleaved caspase 3 (9664), cleaved poly (ADP‐ribose) polymerase (PARP) (5625), E-cadherin (3195), N-cadherin (13116), and SQSTM1/p62 (8025) were obtained from CST company (NJ, US).
Hoechst 33342 b2261
Manufactured by Merck Group
Sourced in United States
Hoechst 33342 (B2261) is a fluorescent dye used for nucleic acid staining in biological research applications. It binds to the minor groove of DNA and emits blue fluorescence upon excitation. The dye can be used to label and visualize cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Soybean trypsin inhibitor, by Thermo Fisher Scientific (2 mentions)
N acetyl l cysteine nac, by Beyotime (1 mentions)
Apoptosis detection kit 556547, by BD (1 mentions)
Antibodies against β actin 20536 1 ap, by Proteintech (1 mentions)
Bcl xl 10783 1 ap, by Proteintech (1 mentions)
Ab192890, by Abcam (1 mentions)
Ab182858, by Abcam (1 mentions)
Ab133528, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab213524, by Abcam (1 mentions)
Cell counting kit 8 cck 8, by Selleck Chemicals (1 mentions)
Ab76003, by Abcam (1 mentions)
Ab207612, by Abcam (1 mentions)
Ab92536, by Abcam (1 mentions)
A31572, by Thermo Fisher Scientific (1 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
Donkey serum, by Jackson ImmunoResearch (1 mentions)
Dragonfly spinning disc confocal microscope, by Oxford Instruments (1 mentions)
Ab27591, by Abcam (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
12 protocols using hoechst 33342 b2261
1
Thymoquinone-Induced Apoptosis and Autophagy
2
Immunofluorescence Staining of Ovarian Tissue
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Immunostaining was performed according to previously described protocols (60 (link)). Briefly, the collected ovaries were fixed with 4% (w/v) PFA for 12 to 24 hours, dehydrated, embedded in paraffin, and cut into 8-μm sections. After deparaffinizing and gradually hydrating the sections, they were put into 0.01% sodium citrate buffer (pH 6.0) and then subjected to microwave antigen retrieval for 16 min. The sections were blocked with 10% donkey serum (Jackson ImmunoResearch) at room temperature for 1 hour, and then the tissue sections were incubated with primary antibodies overnight at 4°C. Next, the sections were thoroughly washed in PBS, incubated with the secondary antibody at room temperature for 1 hour, then stained with Hoechst 33342 (B2261, Sigma-Aldrich) for 1 min. The main primary antibodies used in immunofluorescence are as follows: goat anti-FOXL2 (1:300 dilution; IMG-3228, Novus Biologicals) and DDX4 (1:300 dilution; ab27591, Abcam). Second antibody dilution: donkey anti-goat Alexa Fluor 488 (1:150 dilution; A11055, Invitrogen) and donkey anti-rabbit Alexa Fluor 555 (1:150 dilution; A31572, Invitrogen). Images were acquired on a Nikon Eclipse Ti digital fluorescence microscope, Leica (DMi8), or Andor Dragonfly spinning-disc confocal microscope. The image data were analyzed by the software ImageJ. Image merging was by using the color-merge channels function in ImageJ.
3
Metabolic Profiling of Stem Cells
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Hexokinase (HK), pyruvate kinase (PK), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) enzyme activity kits were obtained from Jiancheng (Nanjing, China). MitoTracker Red CMXRos (M7512) and Alexa Fluor 488 Phalloidin (A12379) were obtained from Invitrogen (Carlsbad, CA, USA). The XF Cell Energy Phenotype Test Kit, XFp extracellular flux analyzer, XFp culture microplates, and bicarbonate-free DMEM were obtained from Seahorse Bioscience Agilent Technologies (North Billerica, MA, USA). Hoechst 33342 (B2261) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP, C2759) were purchased from Sigma–Aldrich (St Louis, MO, USA). The following antibodies were used: CD90 (FITC, mouse anti-human, BioLegend, 328107), CD105 (PerCP/Cy5.5, mouse anti-human, BioLegend, 323215), CD19 (PE, mouse anti-human, BioLegend, 982402), CD34 (PE, mouse anti-human, BioLegend, 343505), AMPK (Abcam, ab80039), p-AMPK (Abcam, ab133448), anti-GAPDH (Abcam, ab9485,ab8245), goat anti-rabbit IgG (H + L)-HRP (Bio-Rad Laboratories, 1706515), goat anti-mouse IgG (H + L)-HRP (Bio-Rad Laboratories,1706516) and goat anti-rabbit IgG (H+L) Alexa Fluor 594-conjugated secondary antibody (Invitrogen, R37117).
4
Soybean Trypsin Inhibitor for Keratinocyte Culturing
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Soybean Trypsin Inhibitor (17,075–029), TrypLE and Defined Keratinocyte SFM (DKSFM) were from Gibco. Dulbecco's Modification of Eagle's Medium (DMEM; 10–017-CV) was purchased from Corning. Collagen (Col) IV (354,233) was from BD Biosciences. ESGRO Leukemia Inhibitory Factor (LIF) (10^7 U/ml, ESG1107), retinoic acid (RA; 302–79-4) and Hoechst 33,342 (B2261) were from Sigma; recombinant mouse bone morphogenetic protein-4 (BMP4; 120-05ET) was from PEPROTECH. Dioxin, i.e. TCDD, was from AccuStandard and dissolved in dimethyl sulfoxide (DMSO; 67–68-5, Sigma). The rest cell culture media and reagents, and antibodies are listed in Supplementary Table s2 and s3 .
5
Fluorescence Analysis of Cell Death
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Cell death was assessed by staining with Hoechst 33342 (B-2261, Sigma-Aldrich, St. Louis, MO, USA) and propidium iodide (PI, P4170, Sigma-Aldrich) followed by fluorescence microscopy (Zeiss LSM710; Carl Zeiss, Oberkochen, Germany). Briefly, RSC96 cells were plated at an initial density of 1 × 104 cells/well in 24-well plates. After treatments, the nuclei were stained by Hoechst 33342 (10 μM) for 15 min at room temperature, and then dead cells were stained with PI (5 μg/mL) for 15 min in the dark. The cells were observed immediately by Zeiss LSM710 microscope, capturing six random fields for each group. Cell death was quantitated as the percentage of PI-positive cells relative to the total cell number (Hoechst 33342-positive cells). All experiments were performed at least three times.
6
Soybean Trypsin Inhibitor Assay Protocol
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Soybean Trypsin Inhibitor (17075–029) was from Gibco. CaCl2 solution (C-34006) was from PromoCell; Collagen IV (354233) was from BD Biosciences Discovery; calcium concentration kit (MAK022-1KT) and Hoechst 33342 (B2261) were from Sigma. All antibodies, cell culture media and reagents are listed in S1 and S2 Tables in S2 File .
7
High-Throughput Cell Viability Assay
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We seeded 4000 proliferating or 10,000 senescent cells per well in CostarAssay 96‐well plates (Corning 3904, Sigma‐Aldrich). The day after seeding, we treated cells with compounds of interest at various concentrations with at least three wells per condition (technical triplicates). In negative control wells, we treated cells with vehicle. In baseline control wells, we treated cells with 20 µM etoposide which very rapidly stops proliferation (Carvalho et al., 2019 (link)). The number of cells in these wells at the end of the incubation period was used as a surrogate for the initial cell number at the time of drugs addition and used for normalization. Cells were fixed and their nuclei stained by incubation in 1% paraformaldehyde (P6148, Sigma‐Aldrich), 0.1% Triton X‐100 (T8787, Sigma‐Aldrich), and 10 µg/ml Hoechst 33342 (B2261, Sigma‐Aldrich) in PBS, for 30 min at room temperature, then washed with PBS. Images were acquired on a CellInsight CX5 (Thermo Fisher Scientific) screening microscope with a 4× objective or on an Operetta (Perkin‐Elmer) screening microscope with a 10× objective. Surviving cell numbers in each well were determined by automated nucleus segmentation and counting.
8
Multicolor Immunofluorescence Imaging of Tumor
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Five µm frozen section of tumor samples were permeabilized with ethanol-acetone (19v–1v) before saturation in PBS- BSA5%. Sequential staining with anti-CD31/anti-MMP9 antibodies or anti-CD45/anti-MMP9 antibodies were done (CD31, JC70A, Dako, 1/50—CD45, HI30, Invitrogen, 1/200—MMP9, ab38898 Abcam, 1/1000). Secondary antibodies were purchased from Molecular Probes™, goat anti mouse-AlexaFluor 488 (A11001) for antibodies targeting CD31 and CD45 and goat anti rabbit-AlexaFluor 568 (A11011) for anti-MMP9 antibody. Nuclei were stained with Hoechst 33,342 (B2261, Sigma, 1/1000). Fluorescent microcopy pictures were done with Zeiss® Observer. Z1 and ZenPRO software and analysis were done with ImageJ software.
9
AMPK Signaling Pathway Antibody Validation
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All chemicals and agents used in this study were purchased from Sigma-Aldrich and Merck (St. Louis, MO) unless otherwise indicated. Anti-rabbit AMPK antibody was purchased from Abcam (ab32047, Cambridge, UK); Alexa Fluor 488 goat anti-rabbit antibody was purchased from Invitrogen (Carlsbad, CA, USA). Hoechst 33,342 (B2261) were purchased from Sigma-Aldrich and Merck (St. Louis, MO, USA). Rabbit anti-β-actin antibody (3700) were purchased from Cell Signaling Technology (Devers, MA, USA).
10
Analyzing ER Stress Signaling Pathways
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4-PBA (P21005) and Hoechst 33342 (B2261) were purchased from Sigma-Aldrich (St. Louis, MO, United States), dissolved in dimethyl sulfoxide (DMSO) and stored at -20°C before use. TM (TM, ab120296) and antibodies against GRP78 (ab21685), IRE1 (ab37073), and phospho-IRE1 (Ser724, ab48187) were obtained from Abcam (Cambridge, MA, United States). Antibodies against microtubule-associated protein 2 (MAP2, #4542), eukaryotic translation initiation factor 2α (eIf2α, #5324), phospho-eIf2α (Ser51, #3398), PERK (#3192), phospho-PERK (Thr980, #12185), Bax (#2772), Bcl-2 (#2870), Cytochrome C (Cyto C, #4280), Bad (#9292), phospho-Bad (Ser112, #9291), PARP (#9532), and β-Actin (#3700) were purchased from Cell Signaling Technology (Beverly, MA, United States). Antibody against ATF6 (IMG273) was purchased from Novus Biologicals (Littleton, CO, United States).
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