Hoechst 33342 trihydrochloride trihydrate

Manufactured by Thermo Fisher Scientific

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Hoechst 33342 trihydrochloride trihydrate is a fluorescent dye used in biological research. It binds to DNA and emits blue fluorescence when excited by ultraviolet light. The compound is often used for staining nuclei and for cell cycle analysis.

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Lab products found in correlation

Bovine serum albumin (bsa), by Merck Group (5 mentions) Whatman, by Cytiva (4 mentions) Prolong diamond antifade mountant, by Thermo Fisher Scientific (4 mentions) Triton x 100, by Merck Group (4 mentions) Paraformaldehyde (pfa), by Merck Group (3 mentions) Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (2 mentions) Anti flag m2, by Merck Group (2 mentions) Anti iκbα, by Cell Signaling Technology (2 mentions) L glutamine, by Corning (2 mentions) Fetal bovine calf serum, by Corning (2 mentions) Fitc labeled tomato lectin fl 1171 1, by Vector Laboratories (2 mentions) Dspe peg2000, by Avanti Polar Lipids (2 mentions) Egg phosphatidylethanolamine, by Avanti Polar Lipids (2 mentions) Anti mouse cd45, by BioLegend (2 mentions) Lsm 700, by Zeiss (2 mentions) Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Merck Group (2 mentions) Phalloidin cruzfluo 555 conjugate, by Santa Cruz Biotechnology (2 mentions) Dmi8 microscope, by Leica (2 mentions) Aaj19943k2, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

Hoechst 33342 (7040 citations) Hoechst (1591 citations) Hoechst 33342 trihydrochloride (25 citations) Trihydrochloride (11 citations) Hoechst 33342 (Hoechst) (5 citations) Hoechst trihydrochloride trihydrate (2 citations)

54 protocols using hoechst 33342 trihydrochloride trihydrate

Characterization of Iohexol Nanoparticles

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All the chemicals were obtained from Sigma Aldrich (St Louis, MO, USA) and used without further purification, unless otherwise specified. Iohexol (Omnipaque™) was obtained from GE Healthcare (UK). MitoTracker® Red CMXRos, Image-iT®-DEAD Green™ viability, HCS Cell Mask™ deep red and Hoechst 33342 (trihydrochloride, trihydrate) were purchased from Life Technologies (Oregon, USA). Sucrose was acquired from VWR International Ltd (England, UK) and paraformaldehyde solution 37–41% was acquired from Fisher Scientific (England, UK). A spectrum 100 spectrometer (PerkinElmer) was used to perform IR measurements. Dynamic light scattering (DLS) and Z-potential were measured using a Zetasizer Nanoseries spectrophotometer (Malvern instruments) at 25 °C. TEM samples were prepared on carbon-coated copper grids (200 mesh, Agar scientific) and TEM images were acquired using a Tecnai T20 instrument (FEI). Powder-XRD data were obtained on a Bruker D8 Advance powder diffractometer with a Cu Kα X-ray source (λ = 1.54058 Å) operating at 40 kV and 40 mA and a Sol-X detector.

Imberti C., Fedatto Abelha T., Yan Y., Lange J., Cui X., Szanda I., Goh V., Dailey L.A, & T. M. de Rosales R. (2020). Synthesis and in vivo evaluation of PEG-BP–BaYbF5 nanoparticles for computed tomography imaging and their toxicity. Journal of Materials Chemistry. B, 8(34), 7723-7732.

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Eosinophil GPR120 Immunofluorescence

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Eosinophils, seeded in 8-well Millicell EZ slides (Millipore, Billerica, MA), were incubated for 5 min in 0.1% bovine serum albumin (BSA) and phorbol 12-myristate 13-acetate (1 ng/ml, Sigma-Aldrich, St Louis, MO) to adhere to the slide and then fixed with 3% paraformaldehyde for 10 min. The slides were washed with phosphate buffered saline (PBS), incubated with blocking buffer (1% BSA PBS) overnight, and incubated with rabbit GPR120 antibody (1:100, Bioworld Technology, Inc., St. Louis Park, MN) or normal rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 120 min. Next, the slides were incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:200, Life Technologies Corporation) for 30 min. To visualize nuclei, slides were stained with Hoechst 33342 trihydrochloride trihydrate (Life Technologies Corporation). The slides were analyzed using a confocal microscope (LSM510; Carl Zeiss Co., Ltd, Jena, Germany).

Konno Y., Ueki S., Takeda M., Kobayashi Y., Tamaki M., Moritoki Y., Oyamada H., Itoga M., Kayaba H., Omokawa A, & Hirokawa M. (2015). Functional Analysis of Free Fatty Acid Receptor GPR120 in Human Eosinophils: Implications in Metabolic Homeostasis. PLoS ONE, 10(3), e0120386.

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Analysis of AnkA Protein Localization in HEK293T Cells

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pCMV-Tag2b-ankA or ankA truncation plasmids were transfected into HEK293T cells. 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton-X in PBS. Rinsed cells were incubated with anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO) for 1 h, washed 4 times with 0.2% Tween-20 in PBS, and incubated with AlexaFluor488 goat anti-mouse secondary antibody (Life Technologies, Coralville, IA) for 1 h. Nuclei were stained with 1:5000 dilution of Hoechst 33342, trihydrochloride, trihydrate (Life Technologies, Coralville, IA) and washed again 4 times with 0.2% Tween-20 in PBS. For confocal microscopy, cells were viewed using a Zeiss LSM 510 Meta Confocal Microscope (University of Maryland, Baltimore Confocal Core Facility).

Rennoll-Bankert K.E., Garcia-Garcia J.C., Sinclair S.H, & Dumler J.S. (2015). Chromatin-bound bacterial effector AnkA recruits HDAC1 and modifies host gene expression. Cellular microbiology, 17(11), 1640-1652.

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Zebrafish Larvae Histological Analysis

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Zebrafish larvae were collected at 6 dpf and fixed in 4% paraformaldehyde. Larvae were cryoprotected using sucrose gradients and embedded in OCT. 10μm sections were cut and warmed for 2 h. Sections were stained using a standard protocol.^{13 (link)} (Antibodies: [1:1000] MTCO1 antibody (abcam 14,705) and [1:500] Tag-FP antibody (FluoTag-X2 anti-TagFP Alexa Fluor 647 N0502-AF647-L)).
TUNEL assay was performed using EMD Millipore ApopTag Fluorescein In Situ Apoptosis Detection Kit (S71100) with the following modifications: hydrated sections with IHC PBS (pH 7.4) for 5 min, permeabilized with 20 μg/mL Proteinase K for 5 min, 5 min IHC PBS (pH 7.4) wash, 5 min incubation with equilibration buffer, 2 h TdT enzyme solution incubation, 2 washes with Stop/Wash buffer followed by IHC PBS (pH 7.4) wash, and 1 h Anti-Digoxigenin Conjugate incubation. Nuclei were stained using Hoechst 33,342, trihydrochloride trihydrate (10 μM; Life Technologies, H3570). Images collected on Leica SP8 (RRID: SCR_018169).

Freeman-Cook L.L., Sherman J.M., Brachmann C.B., Allshire R.C., Boeke J.D, & Pillus L. (1999). The Schizosaccharomyces pombe hst4+ Gene Is a SIR2 Homologue with Silencing and Centromeric Functions. Molecular Biology of the Cell, 10(10), 3171-3186.

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Immunofluorescence Staining of Synchronized MEFs

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Synchronized MEFs were trypsinized and plated at a density of 3×10⁵ cells/ml on glass coverslips treated with 0.1% gelatin in standard MEF media containing serum. After 24 hours, cells were fixed with 4% v/v paraformaldehyde in PBS. For sequential immunolabeling, cells were washed with PBS, permeabilized and blocked with 0.1% Triton X-100 and 5% FBS respectively. Primary and secondary antibody incubations were one hour at room temperature followed by 10 min. incubation in Hoechst 33342 trihydrochloride trihydrate (Life Technologies, Molecular Probes, H1399) to counterstain DNA. Primary antibodies were anti-α tubulin (Abcam Ab18251) and anti-centromere proteins (derived from CREST patients, Antibodies Inc., 15-234) and both were used for immunofluorescence at a dilution of 1:200. Coverslips were mounted on microscope slides overnight using Vectashield hard-set mounting medium (Vector Laboratories, H-1400), and imaged on a Leica SP5 confocal microscope or a Zeiss AxioImager epifluorescence microscope.

Czechanski A., Kim H., Byers C., Greenstein I., Stumpff J, & Reinholdt L.G. (2015). Kif18a is specifically required for mitotic progression during germ line development. Developmental biology, 402(2), 253-262.

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Immunocytochemistry of Differentiated iNPC-As

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Ten thousand iNPC‐As were plated per well in 96‐well plates at day 6 of differentiation and fixed 24 h after seeding with 4% paraformaldehyde (PFA) for 10 min. Cells were washed three times with phosphate‐buffered saline (PBS) following which blocking (PBS, 5% horse serum and 0.05% Triton X‐100) was applied for 1 h. All primary antibodies were diluted in blocking solution and their dilution and suppliers are listed in Table S6. Incubation of the primary antibody was performed overnight at 4°C. The next day, cells were washed three times in PBS before the secondary antibody (Table S7) in blocking solution was applied for 1 h at room temperature. Hoechst (Hoechst 33342, Trihydrochloride, Trihydrate, Life tech) diluted 1:6000 was added for 5 min to visualise the nucleus. Cells were then washed twice with PBS and imaged using the Opera Phenix high‐content imager (PerkinElmer).

Gatto N., Dos Santos Souza C., Shaw A.C., Bell S.M., Myszczynska M.A., Powers S., Meyer K., Castelli L.M., Karyka E., Mortiboys H., Azzouz M., Hautbergue G.M., Márkus N.M., Shaw P.J, & Ferraiuolo L. (2020). Directly converted astrocytes retain the ageing features of the donor fibroblasts and elucidate the astrocytic contribution to human CNS health and disease. Aging Cell, 20(1), e13281.

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Antibody Validation for Molecular Analyses

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The following antibodies (Abs) were used for western blotting, immunoprecipitation, qPCR, ELISA, confocal microscopy, immunohistochemistry, flow cytometry, or joint injection: anti-TMEM147 (sc-138814, Santa Cruz Biotechnology), anti-TMEM147 (generated by immunizing rabbit with peptide containing amino acids 121-136 VGARGIEFDWKYIQMSC, Sigma-Aldrich), anti-NF-κB p65 (sc-372, Santa Cruz Biotechnology), anti-phospho-p65 S536 (#3033, Cell Signaling Technology, Tokyo, Japan), anti-phospho NF-κB p65 S529 (ab47395, Abcam, Tokyo, Japan), anti-phospho NF-κB p65 S276 (SAB4504488, Sigma-Aldrich), anti-phospho-stat3 (#9145, Cell Signaling Technology), anti-IκBα (#4814, Cell Signaling Technology), anti-phospho-IκBα (#9246, Cell Signaling Technology), anti-BCR (#3902, Cell Signaling Technology), anti-phospho-BCR Y177 (#3901, Cell Signaling Technology), anti-CK2α (#2656, Cell Signaling Technology), anti-phospho-CK2α (SAB4300628, Sigma-Aldrich), anti-α-tubulin (T5168, Sigma-Aldrich), anti-FLAG M2 (F1804, Sigma-Aldrich), rabbit IgG (#3900, Cell Signaling Technology), anti-rabbit IgG (sc-2004, Santa Cruz Biotechnology), anti-mouse IgG (sc-2314, Santa Cruz Biotechnology), anti-goat IgG (sc-3851, Santa Cruz Biotechnology), Alexa Fluor 488 goat anti-rabbit IgG (H+L) (A11055, Life technologies, Tokyo, Japan) and Hoechst 33342 trihydrochloride trihydrate (H3570, Life technologies).

Ota M., Tanaka Y., Nakagawa I., Jiang J.J., Arima Y., Kamimura D., Onodera T., Iwasaki N, & Murakami M. (2020). Role of Chondrocytes in the Development of Rheumatoid Arthritis Via Transmembrane Protein 147-Mediated NF-κB Activation. Arthritis & rheumatology (Hoboken, N.J.), 72(6).

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Lipid-Based Nanoparticle Trafficking Visualization

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DiD (1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindotricarbocyanine, 4-chlorobenzenesulfonate salt), and DiI (1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindotricarbocyanine Perchlorate) were from Biotium (Hayward, CA, USA) and were stored as 10 mM sock in ethanol. Whatman Nucleopore Track-Etch Membranes, bovine serum albumin, and the chemicals for synthesis were from Sigma-Aldrich (St. Louis, MO, USA). Nitrocellulose membrane (0.45 μm) was from Bio-Rad (Hercules, CA, USA). Egg phosphatidylcholine (EPC), distearoyl phosphatidylethanolamine (DSPE)-PEG2000 were from Avanti Polar Lipids (Alabaster, AL, USA). Nuclear staining reagent Hoechst 33342 trihydrochloride trihydrate was purchased from Life Technologies (Carlsbad, CA, USA). Fetal bovine calf serum, RPMI 1640 growth medium supplemented with L-glutamine were from Corning Inc. (New York, NY, USA). FITC-labeled tomato lectin (FL-1171-1) was from Vector Laboratories (Burlingame, CA, USA). PEGylated liposomal doxorubicin (LipoDox^®, Sun Pharma) was obtained in sterile leftover vials from the University of Colorado Cancer Center pharmacy. Rhodamine B dextran (70kDa) was from ThermoFisher.

Balyasnikova I.V., Zannikou M., Wang G., Li Y., Duffy J.T., Levine R.N., Seblani M., Gaikwad H, & Simberg D. (2022). Indocarbocyanine nanoparticles extravasate and distribute better than liposomes in brain tumors. Journal of controlled release : official journal of the Controlled Release Society, 349, 413-424.

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Antibody Toolkit for Cellular Signaling

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The following antibodies were used: anti-GGT1 (ab88864 for western blotting, Abcam), anti-GTF2I-C-terminal (ab135619 for immunohistochemistry, Abcam), anti-p65 (C-20, Santa Cruz), anti-phospho-p65 (Ser536 93H1, Cell Signaling Technology), anti-IκBα (Cell Signaling Technology), anti-phospho-IκBα (Ser32/36 5A5, Cell Signaling Technology), anti-STAT3 (Cell Signaling Technology), anti-p-STAT3 (Cell Signaling Technology), anti-FLAG M2 (Sigma-Aldrich), anti-c-Myc (Sigma-Aldrich), Alexa Fluor 488 goat anti-rabbit IgG (H + L) (Invitrogen), Alexa Fluor 546 goat anti-mouse IgG (H + L) (Invitrogen) and Hoechst 33,342 trihydrochloride trihydrate (Life Technologies).

Furukawa R., Kuwatani M., Jiang J.J., Tanaka Y., Hasebe R., Murakami K., Tanaka K., Hirata N., Ohki I., Takahashi I., Yamasaki T., Shinohara Y., Nozawa S., Hojyo S., Kubota S.I., Hashimoto S., Hirano S., Sakamoto N, & Murakami M. (2024). GGT1 is a SNP eQTL gene involved in STAT3 activation and associated with the development of Post-ERCP pancreatitis. Scientific Reports, 14, 12224.

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Fluorescent Lipid Labeling of Cells

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DiD (1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindotricarbocyanine, 4-chlorobenzenesulfonate salt), DiI (1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindotricarbocyanine Perchlorate) and DiR (1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindotricarbocyanine Iodide) were from Biotium (Hayward, CA, USA). Cy5-DSPE and Cy3-DSPE were synthesized as described previously. ²⁴ All fluorescent lipids were stored at −20ºC as 4-10mM stocks in ethanol. Whatman Nucleopore Track-Etch Membranes, bovine serum albumin were from Sigma-Aldrich (St. Louis, MO, USA). Hydrogenated soy phosphatidylcholine (HSPC), cholesterol, egg phosphatidylethanolamine, DSPE-PEG2000 were from Avanti Polar Lipids (Alabaster, AL, USA) and were kept as chloroform stocks at −20ºC. Rabbit anti-mouse CD81 (Cat. 10037) was from Cell Signaling Technology (Danvers, MA, USA). Rat anti-mouse CD31 (Cat. 102402) was from BioLegend (San Diego, CA). Goat anti-rabbit Alexa 647 antibody was from ThermoFisher. Hoechst 33342 trihydrochloride trihydrate was purchased from Life Technologies (Carlsbad, CA, USA). FITC-labeled tomato lectin (Cat. FL-1171-1) was from Vector Laboratories (Burlingame, CA, USA).

Li Y., Lofchy L., Wang G., Gaikwad H., Fujita M, & Simberg D. (2022). PEGylated liposomes accumulate in the areas relevant to skin toxicities via passive extravasation across “leaky” endothelium. ACS nano, 16(4), 6349-6358.

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