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Hoechst reagent

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The Hoechst reagent is a fluorescent dye used in laboratory settings for the detection and quantification of DNA. It binds to the minor grooves of double-stranded DNA, emitting fluorescence upon excitation. The Hoechst reagent is commonly used in various applications, such as cell cycle analysis, apoptosis detection, and nucleic acid quantification.

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Lab products found in correlation

Sea block blocking solution, by Thermo Fisher Scientific (2 mentions) Rabbit anti gfp, by Abcam (2 mentions) Anti rat igg, by Thermo Fisher Scientific (2 mentions) Rat anti cadn ex 8, by Developmental Studies Hybridoma Bank (2 mentions) Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (2 mentions) Tcs sp2, by Leica camera (2 mentions) Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions) Matrigel, by Corning (1 mentions) Fluoromount g, by Thermo Fisher Scientific (1 mentions) Tcs sp5, by Leica camera (1 mentions) Tcs sp8, by Leica camera (1 mentions) 5 3h d glucose, by PerkinElmer (1 mentions) Axio imager a1, by Zeiss (1 mentions) Phalloidin atto 488, by Merck Group (1 mentions) Draq5, by LI COR (1 mentions) Odyssey imager, by LI COR (1 mentions) Celltiter glo, by Promega (1 mentions) Draq5, by Biostatus (1 mentions) Dm4000b, by Leica camera (1 mentions) Alexa fluor 647 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)

11 protocols using hoechst reagent

1

Quantifying Neuronal Viability Using Multimodal Assays

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We used three assays for structure and function to quantify viability in a blind and unbiased manner: the infrared DRAQ5 stain for nuclei, infrared immunocytochemistry for the neuronal marker microtubule associated protein 2 (MAP2), and a luminescent assay for ATP levels, as previously described (Posimo et al. 2013 (link), Posimo et al. 2014 (link)). For high-magnification visualization of neurons, synapses, and astrocytes, olfactory bulb cultures were immunostained with mouse anti-MAP2 in conjunction with rabbit anti-synaptophysin or rabbit anti-glial fibrillary acidic protein (GFAP). Antibodies are listed in Supplementary Tables 1 and 2. Antibody binding was visualized with fluorescent secondary goat/donkey anti-mouse IgGs (488 nm) and secondary goat anti-rabbit IgGs (555 nm). Nuclei were stained with the Hoechst reagent (Sigma-Aldrich). As a negative control for the immunostaining, primary antibodies were omitted in all assays to ensure loss of fluorescent signal. Two blinded observers counted MAP2+ and Hoechst+ profiles from images captured on an epifluorescent microscope (EVOS, Life Technologies).
Crum T.S., Gleixner A.M., Posimo J.M., Mason D.M., Broeren M.T., Heinemann S.D., Wipf P., Brodsky J.L, & Leak R.K. (2015). Heat shock protein responses to aging and proteotoxicity in the olfactory bulb. Journal of neurochemistry, 133(6), 780-794.
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2

Immunofluorescence Characterization of iPSC-derived Cells

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iPSC-derived LM and NC progenitors or differentiated SMCs were plated on glass coverslips coated with Matrigel (Corning) and 24 h later fixed with ice-cold 90% ethanol. Fixed cells were rehydrated with 1 × PBS, permeabilized with 0.2% Triton solution in 1 × PBS and non-specific binding diminished using Sea Block Blocking Solution (Thermo Scientific). Cells were incubated with primary antibodies (all purchased from Abcam, Burlingame, CA) targeting the following markers: (i) myocyte enhancer factor-2c (MEF2c) and homeobox protein NKX2.5 (LM progenitor cells); (ii) Nestin (NC progenitor cells); (iii) Calponin 1 (CNN1) and Transgelin (TGLN) (differentiated SMCs); or (iv) immunoglobulin isotype control, then stained with the appropriate Alexa Fluor secondary antibodies (Thermo Fisher Scientific). Preps were counter-stained for chromatin with Hoechst reagent (1 μg/mL) (Sigma-Aldrich). Fluorescent images were captured using a Leica DM5500B Upright Microscope and Photometrics CoolSNAP HQ2 CCD camera, using HC Plan Apo 25-mm objectives.
Iosef C., Pedroza A.J., Cui J.Z., Dalal A.R., Arakawa M., Tashima Y., Koyano T.K., Burdon G., Churovich S.M., Orrick J.O., Pariani M, & Fischbein M.P. (2020). Quantitative proteomics reveal lineage-specific protein profiles in iPSC-derived Marfan syndrome smooth muscle cells. Scientific Reports, 10, 20392.
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3

Proliferative Effects of HB-EGF on MIN6 Cells

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MIN6 cells (passages 25–30) were cultured as previously described (11 (link)). Cells were seeded in six-well plates at a density of 500,000 cells/well. The following day, the cells were cultured in complete medium with increasing concentrations of HB-EGF in the absence or presence of inhibitors for an additional 24 h. For cell counting, cells were fixed with 10% formaldehyde, nuclei were stained by Hoechst reagent (Sigma-Aldrich), and 10 images per well were taken randomly. Cell numbers were determined using ImageJ software (National Institutes of Health).
Zarrouki B., Benterki I., Fontés G., Peyot M.L., Seda O., Prentki M, & Poitout V. (2014). Epidermal Growth Factor Receptor Signaling Promotes Pancreatic β-Cell Proliferation in Response to Nutrient Excess in Rats Through mTOR and FOXM1. Diabetes, 63(3), 982-993.
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4

Immunohistochemical Analysis of Embryos

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Embryos were fixed and immunohistochemically analyzed as described by Schäfer et al. (2007) (link). The following antibodies were used at the noted dilutions: rat anti-CadN-Ex#8 (Iwai et al., 1997 (link)) 1:50 (Developmental Studies Hybridoma Bank), guinea pig anti-β3Tubulin (Buttgereit et al., 1996 (link); Leiss et al., 1988 (link)) 1:10,000, rabbit anti-β-Gal 1:5000 (Biotrend), rabbit anti-GFP 1:500 (abcam ab5665). Primary antibodies were detected using the fluorescent labeled antibodies Alexa-Fluor-488, 568- or 647-conjugated anti-guinea pig, anti-rabbit and anti-rat IgG at a dilution of 1:500 (Invitrogen). DNA was stained with Hoechst reagent (5 g/ml; Sigma-Aldrich), and F-actin was stained with Alexa-Fluor-647–phalloidin (1:100, Invitrogen). For all stainings, specimens were embedded in Fluoromount-G™ (Thermo Fisher Scientific) and observed under a Leica TCS Sp2, TCS Sp5 or TCS Sp8 confocal microscope.
Lübke S., Braukmann C., Rexer K.H., Cigoja L., Rout P, & Önel S.F. (2024). The Abl-interactor Abi suppresses the function of the BRAG2 GEF family member Schizo. Biology Open, 13(1), bio058666.
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5

Analysis of Glycolysis in WT and G6pc2 KO Islets

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Glycolysis was analyzed as described (Ashcroft, et al. 1972 (link); Tamarit-Rodriguez, et al. 1998 (link); Wang and Iynedjian 1997 (link)). Briefly, aliquots of ~100 WT and germline G6pc2 KO islets were incubated for 2 hrs at 37°C in Krebs bicarbonate buffer containing 5.6 mM glucose spiked with 1 μl D-[5-3H]glucose (Perkin Elmer, Waltham, MA; 10Ci/mmol; 1 mCi/ml) in a volume of 100 μl. Following the incubation, islets were pelleted by centrifugation. The supernatant was retained for analysis of 3H2O generation whereas DNA content of the cell pellet was measured using the Hoechst reagent (Sigma, St. Louis, MO) as described (Ashcroft et al. 1972 (link)).
Bosma K.J., Rahim M., Singh K., Goleva S.B., Wall M.L., Xia J., Syring K.E., Oeser J.K., Poffenberger G., McGuinness O.P., Means A.L., Powers A.C., Li W.H., Davis L.K., Young J.D, & O’Brien R.M. (2020). Pancreatic Islet Beta Cell-Specific Deletion of G6pc2 Reduces Fasting Blood Glucose. Journal of molecular endocrinology, 64(4), 235-248.
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6

Immunofluorescence Imaging of HeLa Cells

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HeLa cells were grown overnight in chamber slides with 1.7 cm2. Cells were transfected as described above and incubated for 48 h. Next, cell culture medium was removed and cells were fixed with 4% PFA for 10 min at RT. Cells were permeabilized with PBS, 0.5% Triton X-100 and blocked with blocking buffer (2% BSA + 0.05% Triton X-100 in PBS). Actin and nuclei were stained by treatment with Phalloidin-Atto 488 (Sigma Aldrich, Buchs, Switzerland) for 1 h and with Hoechst reagent (Sigma Aldrich, Buchs, Switzerland) for 5 min. Slides were mounted with Mowiol/DABCO solution (Sigma Aldrich, Buchs, Switzerland) and visualized using a Zeiss Axio Imager A1 fluorescence microscope equipped with a HXP 120 Laser for fluorophore excitation. The experiments were performed in triplicate.
Sabino F., Madzharova E, & auf dem Keller U. (2020). Cell density-dependent proteolysis by HtrA1 induces translocation of zyxin to the nucleus and increased cell survival. Cell Death & Disease, 11(8), 674.
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7

Actin Cytoskeleton Dynamics in PECs

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NTC‐, IKKα‐ and IKKβ siRNA‐treated neonatal PECs were plated on glass coverslips 24 hours after transfection and starved for 12 hours. Starvation media (SM) was replaced with either new SM (FBS 0.2%, complete EGM (FBS 5% with growth factors), or SM with VEGF (50 ng/mL). Fixed cells were rehydrated, permeabilized and non‐specific binding diminished using Sea Block Blocking Solution (Thermo Scientific). Cells were then incubated with Phalloidin‐TRITC reagent (ThermoFisher Scientific) and counter‐stained for chromatin with Hoechst reagent (1 μg/mL) (Sigma‐Aldrich). Fluorescent images were captured using a Leica DM5500B Upright Microscope and Photometrics CoolSNAP HQ2 CCD camera, using HC Plan Apo 25‐mm objectives at ×40 magnification.
Iosef C., Liu M., Ying L., Rao S.P., Concepcion K.R., Chan W.K., Oman A, & Alvira C.M. (2018). Distinct roles for IκB kinases alpha and beta in regulating pulmonary endothelial angiogenic function during late lung development. Journal of Cellular and Molecular Medicine, 22(9), 4410-4422.
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8

Evaluating Proteasome Inhibitor Effects on Cell Viability

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N2a cells were pretreated with the proteasome inhibitor MG132 at an IC50 concentration (2 μM for the ATP assay and 1 μM for the cell number assay) in the absence or presence of a range of MI-4 concentrations (0.05 – 6.4 μM) dissolved in DMSO. Control cells were treated with DMSO. Two days later, viability was assessed by measuring ATP (Cell Titer Glo, Promega). On parallel plates, cells were stained with the nuclear stain DRAQ5 (Biostatus) in conjunction with the cytosolic stain Sapphire (LI-COR) or the Hoechst reagent (Sigma-Aldrich), as previously published (Posimo et al., 2014 (link)). Infrared DRAQ5+Sapphire signal was quantified on an Odyssey Imager (LI-COR) and Hoechst-stained cells were photographed on an EVOS epifluorescent microscope (Life Technologies).
Talbot J.N., Geffert L.M., Jorvig J.E., Goldstein R.I., Nielsen C.L., Wolters N.E., Amos M.E., Munro C.A., Dallman E., Mereu M., Tanda G., Katz J.L., Indarte M., Madura J.D., Choi H., Leak R.K, & Surratt C.K. (2016). Rapid and sustained antidepressant properties of an NMDA antagonist/monoamine reuptake inhibitor identified via transporter-based virtual screening. Pharmacology, biochemistry, and behavior, 150-151, 22-30.
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9

Collagen I and MMP2 Immunostaining in Aortic Sections

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Aortic cross sections (7 μm) were incubated with primary antibodies against collagen I and Mmp2 (Abcam) overnight at 4°. Incubation with secondary antibodies (Alexa Fluor 647 goat anti-rabbit; Molecular Probes) was performed for 1 h. Nuclear counterstaining was performed with Hoechst reagent (bisBenzide H 33258; Sigma-Aldrich, St. Louis, MO, USA). Negative controls were performed with the omission of the primary antibody. Imaging was performed using a Leica DM4000B (Buffalo Grove, IL, USA).
Schellinger I.N., Wagenhäuser M., Chodisetti G., Mattern K., Dannert A., Petzold A., Jakubizka-Smorag J., Emrich F., Haunschild J., Schuster A., Schwob E., Schulz K., Maegdefessel L., Spin J.M., Stumvoll M., Hasenfuß G., Tsao P.S, & Raaz U. (2021). MicroRNA miR-29b regulates diabetic aortic remodeling and stiffening. Molecular Therapy. Nucleic Acids, 24, 188-199.
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10

Immunohistochemical Analysis of Embryos

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Embryos were fixed and immunohistochemically analyzed as described by Schäfer et al. (2007) . The following antibodies were used at the noted dilutions: rat anti-CadN-Ex#8 (Iwai et al., 1997) 1:50 (Developmental Studies Hybridoma Bank), guinea pig anti-3Tubulin (Buttgereit et al., 1996; Leiss et al., 1988) 1:10,000, rabbit anti- -Gal 1:5000 (Biotrend), rabbit anti-GFP 1:500 (abcam). Primary antibodies were detected using the fluorescent-labeled antibodies Alexa-Fluor-488, 568-or 647-conjugated anti-guinea pig, anti-rabbit and anti-rat IgG at a dilution of 1:500 (Invitrogen). DNA was stained with Hoechst reagent (5 g/ml; Sigma-Aldrich), and F-actin was stained with Alexa-Fluor-647-phalloidin (1:100, Invitrogen). For all stainings, specimens were embbeded in Fluoromount-G TM (Thermo Fisher Scientific) and observed under a Leica TCS Sp2 or TCS Sp8 confocal microscope.
Lübke S., Braukmann C., Rexer K., Cigoja L., & Önel S. (2020). The Abl-interactor Abi suppresses the function of the BRAG2 GEF family member Schizo.
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