Bisbenzimide hoechst

Manufactured by Merck Group

Sourced in France, United States

BisBenzimide Hoechst is a fluorescent dye used for staining and visualizing nucleic acids, particularly DNA, in biological samples. It binds to the minor groove of DNA, emitting a blue fluorescence when exposed to ultraviolet (UV) or blue light.

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Lab products found in correlation

Bovine serum albumin (bsa), by Merck Group (3 mentions) Methanol, by Thermo Fisher Scientific (2 mentions) Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions) Rabbit anti human zo 1, by Thermo Fisher Scientific (1 mentions) Mouse anti nestin, by Novus Biologicals (1 mentions) Mouse anti sma, by Agilent Technologies (1 mentions) Mouse anti afp, by Merck Group (1 mentions) Alexa fluor 594 donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions) Alexa fluor 488 donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions) Donkey serum, by Merck Group (1 mentions) Acetone, by Carl Roth (1 mentions) Primary mouse monoclonal nis specific antibody, by Merck Group (1 mentions) Methanol, by Carl Roth (1 mentions) Alexa fluor 488 or 568 secondary antibodies, by Thermo Fisher Scientific (1 mentions) Lsm 510, by Leica camera (1 mentions) Vectashield, by Agilent Technologies (1 mentions) Goat anti mouse igg alexa546, by Thermo Fisher Scientific (1 mentions) Alexa fluor 546 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions) Dako fluorescent mounting media, by Agilent Technologies (1 mentions) Triton x 100, by Merck Group (1 mentions)

9 protocols using bisbenzimide hoechst

iPSC-derived RPE Characterization and Differentiation

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For the RPE characterization studies, iPSC-derived RPE was seeded and processed as previously described (19). For the differentiation assay, embryoid bodies were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and blocked in 10% donkey serum (Millipore) and 1% BSA (Sigma-Aldrich). Primary antibodies were diluted in 1% donkey serum and 1% BSA, and incubated overnight at 4 °C. Secondary antibodies were incubated 45 min at room temperature with 0.2 µg/ml bisBenzimide Hoechst (Sigma-Aldrich) prior to mounting in ProLong Diamond Antifade Mountant (Molecular probes, Life Technologies). Primary antibodies: 1:100 dilution rabbit anti-human ZO-1 (Invitrogen, Life technologies), 1:250 rabbit anti-human LRAT (Abcam), 1:500 mouse anti-human Bestrophin-1 (Abcam), 1:200 mouse anti-Nestin (Novus Biologicals, Lille, France), 1:200 mouse anti-SMA (Dako, Les Ulis, France) and 1:200 mouse anti-AFP (Sigma-Aldrich). Secondary antibodies: 1:500 dilution donkey anti-rabbit IgG-Alexa Fluor 594, and donkey anti-mouse IgG-Alexa Fluor 488 (Jackson ImmunoResearch). Images were taken using a Zeiss ApoTome 2 Upright wide-field microscope (Carl Zeiss SAS).

Erkilic N., Gatinois V., Torriano S., Bouret P., Sanjurjo-Soriano C., De Luca V., Damodar K., Cereso N., Puechberty J., Sanchez-Alcudia R., Hamel C.P., Ayuso C., Meunier I., Pellestor F, & Kalatzis V. (2019). A Novel Chromosomal Translocation Identified due to Complex Genetic Instability in iPSC Generated for Choroideremia. Cells, 8(9), 1068.

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Immunofluorescence Staining of NIS in HSP70B-MSCs

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HSP70B-NIS-MSCs were seeded directly on FBS-coated microscope slides and grown until 60% confluent. 6 h after hyperthermia, the slides were air-dried overnight at room temperature and monolayers fixed with 80% methanol (Carl Roth) for 5 min at 4 °C, followed by 100% acetone (Carl Roth) for 2 min at -20 °C. Following blocking with 12% bovine serum albumin (Sigma-Aldrich) in phosphate- buffered saline (PBS; Sigma Aldrich) for 30 min, cells were then incubated with a primary mouse monoclonal NIS-specific antibody (Merck Millipore; dilution 1:500) for 90 min. A secondary Cy3 AffiniPure donkey anti-rabbit IgG antibody (Jackson ImmunoResearch; dilution 1:400) and bisbenzimide (Hoechst; Sigma Aldrich; dilution 1:2000) to counterstain nuclei were added for 30 min. Pictures were taken using an Axiovert 135 TV fluorescence microscope in combination with an AxioCam MRm CCD camera and the AxioVision Rel. 4.8 software (Carl Zeiss Microscopy GmbH, Jena, Germany).

Tutter M., Schug C., Schmohl K.A., Urnauer S., Schwenk N., Petrini M., Lokerse W.J., Zach C., Ziegler S., Bartenstein P., Weber W.A., Wagner E., Lindner L.H., Nelson P.J, & Spitzweg C. (2020). Effective control of tumor growth through spatial and temporal control of theranostic sodium iodide symporter (NIS) gene expression using a heat-inducible gene promoter in engineered mesenchymal stem cells. Theranostics, 10(10), 4490-4506.

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Cell Phenotype Characterization by Immunofluorescence

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To characterize cell phenotype, fixed cells were permeabilized with ice-cold methanol (Thermo Fisher Scientific) for 20 min and blocked for non-specific binding with 10% NGS (Chemicon International, Merck Millipore) in PBS for 2 h. After that, cells were incubated with primary antibody diluted in 5% NGS in PBS for 24 h at +4°C. Then, the cells were washed 3 times with PBS and incubated with Alexa-Fluor-488 or -568 secondary antibodies (Molecular Probes, Invitrogen) diluted in 5% NGS in PBS for 2 h at RT and protected from light. Following washing, cell nuclei were stained with 2.5 μg/ml bisBenzimide (Hoechst; Sigma-Aldrich) for 5 min. For BrdU staining, before blocking of non-specific sites the cells were incubated with 2 N HCl for 10 min, washed again and then stained using the protocol described above.

Savchenko E., Singh Y., Konttinen H., Lejavova K., Mediavilla Santos L., Grubman A., Kärkkäinen V., Keksa-Goldsteine V., Naumenko N., Tavi P., White A.R., Malm T., Koistinaho J, & Kanninen K.M. (2017). Loss of Cln5 causes altered neurogenesis in a mouse model of a childhood neurodegenerative disorder. Disease Models & Mechanisms, 10(9), 1089-1100.

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Corneal Immunofluorescence Staining Protocol

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Corneal explants were fixed in 4% paraformaldehyde overnight, transferred to 30% sucrose/PBS solution overnight and embedded in OCT compound (Tissue-Tek, Sakura). Ten-micron-thick transversal frozen sections were cut with a cryo-microtome. Sections were stained with 1 μg/ml bis-benzimide Hoechst (Sigma-Aldrich) and mounted with Vectashield (DAKO). For the immunofluorescence CD34 staining, sections were thawed for 15 min at room temperature, washed in PBS for 15 min to remove OCT, permeabilized (0.1% Triton) for 15 min at room temperature and blocked in 2.5% horse serum/0.5% BSA/0.1% Triton for 30 min. Primary antibodies were incubated on sections overnight at 4 °C, and the secondary antibody incubated 90 min, at room temperature prior to Hoechst labeling and mounting. For the human corneas, the primary antibody used was 1:100 mouse anti-human CD34 (clone B1-3C5; AbCam, Cambridge, UK) and the secondary antibody 1:500 goat anti-mouse IgG-Alexa546 (Molecular probes, Invitrogen). For the dog corneas, the primary antibody used was 1:100 mouse anti-dog CD34-PE (clone 1H6, eBioscience) and the secondary antibody 1:500 goat anti-mouse IgG-Alexa Fluor 546 (Molecular probes, Invitrogen). Observations and acquisitions were performed with either a Zeiss LSM510 or a LEICA SP5 laser scanning confocal microscope. Image analyses were performed using MetaMorph software.

Serratrice N., Cubizolle A., Ibanes S., Mestre-Francés N., Bayo-Puxan N., Creyssels S., Gennetier A., Bernex F., Verdier J.M., Haskins M.E., Couderc G., Malecaze F., Kalatzis V, & Kremer E.J. (2014). Corrective GUSB transfer to the canine mucopolysaccharidosis VII cornea using a helper-dependent canine adenovirus vector. Journal of controlled release : official journal of the Controlled Release Society, 181, 22-31.

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Fibroblast Transfection with GFP Plasmid

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One million CHM3 fibroblasts were nucleofected with 1 µg DNA of the pCHM:EGFP fusion plasmid and 100 µl of P2 Primary Cell 4D-Nucleofector solution (Lonza, Levallois-Perret, France) using the program EN-150 of the Amaxa 4D-Nucleofector according to the manufacturer’s instructions. Non-transfected and transfected cells were then seeded in four 1.9 cm² wells containing glass coverslips. Media was refreshed the following day and, 72 h post-nucleofection, cells were fixed with 4% Alfa Aesar paraformaldehyde (PFA; ThermoFisher Scientific), blocked in 5% FCS and 1% bovine serum albumin (BSA; Sigma-Aldrich), and permeabilised with 0.2% Triton X-100 (Sigma-Aldrich). The coverslips were then incubated with a 1:200 dilution of rabbit anti-GFP antibody (Invitrogen-Molecular probes) overnight at 4 °C and with a 1:1000 dilution donkey anti-rabbit IgG-Alexa Fluor 488 secondary antibody containing 0.2 µg/ml bisBenzimide Hoechst (Sigma-Aldrich) for 45 min at room temperature prior to mounting in Dako Fluorescent Mounting Media (Dako France SAS, Les Ulis, France).

Torriano S., Erkilic N., Baux D., Cereso N., De Luca V., Meunier I., Moosajee M., Roux A.F., Hamel C.P, & Kalatzis V. (2018). The effect of PTC124 on choroideremia fibroblasts and iPSC-derived RPE raises considerations for therapy. Scientific Reports, 8, 8234.

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Embedding Cells in Collagen Gels

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To preserve the relative position of beads on the Fn fibers together with the outgrown cells at the end of the assay, the samples were embedded in collagen gels prior to immunostaining. Briefly, samples were prepared as described for fluorescence microscopy, using unlabeled LEC or HUVEC and Fn fibers. Following a 6h incubation time at 37°C, the samples were fixed with 4% formaldehyde and subsequently covered with 4 mg/ml collagen. The collagen was left to polymerize at 37°C for 20 min and subsequently was washed for 2h with PBS. The samples were additionally fixed for 15min with 4% formaldehyde, washed with 0.1M glycine for 15min and blocked with 10% donkey serum in PBS for 1h. Antibodies used were anti VE-cadherin (R&D Systems) and Alexa Fluor 488 conjugated secondary antibody (Invitrogen). Cell nuclei were counterstained with Hoechst bisbenzimide (Sigma-Aldrich) and the actin cytoskeleton with Alexa Flour 549 conjugated phalloidin (Invitrogen). Stained specimens were examined with a Leica SP5 laser scanning confocal microscope, with a 40x dry APO U-V-I (NA = 0.75) objective lens, using the Leica Application Suite Advanced Fluorescence 2.6.1.7314 software and the images were processed by ImageJ (NIH).

Mitsi M., Schulz M.M., Gousopoulos E., Ochsenbein A.M., Detmar M, & Vogel V. (2015). Walking the Line: A Fibronectin Fiber-Guided Assay to Probe Early Steps of (Lymph)angiogenesis. PLoS ONE, 10(12), e0145210.

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Astrocyte Morphology and Oxidative Damage Analysis

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Morphological changes were analyzed by phase contrast microscopy of astrocyte cultures fixed with 4% paraformaldehyde (Sigma, St Louis, MO, USA) and by confocal imaging after further immunostaining with antiglial fibrillary acidic protein (GFAP, 1:500; Dako, Glostrup, Denmark), followed by Alexa Fluor 546 (1:1000; Molecular Probes, Life Technologies, Alcobendas, Madrid, Spain). Oxidative damage was detected by double immunostaining with antinitrotyrosine (1:50; Abcam, Cambridge, UK), followed by Alexa Fluor 488 (1:1000; Molecular Probes). Cell nuclei were counterstained with Hoechst bisbenzimide (Sigma). Mitochondrial mass was visualized by staining fresh cultures with MitoTracker Green FM (50 nm, 30 min; Molecular Probes). Presence of replicative senescence was analyzed with the Senescence β-Galactosidase Staining kit (Cell Signaling Technology, Danvers, MA, USA), following manufacturer's instructions.

García-Matas S., Paul R.K., Molina-Martínez P., Palacios H., Gutierrez V.M., Corpas R., Pallas M., Cristòfol R., de Cabo R, & Sanfeliu C. (2015). In vitro caloric restriction induces protective genes and functional rejuvenation in senescent SAMP8 astrocytes. Aging Cell, 14(3), 334-344.

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DNA-selective fluorescent dye staining

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The DNA-selective fluorescent dyes SYBR® Green (Invitrogen, Carlsbad, CA, USA; 1:10,000 in PBS) or Hoechst (Bisbenzimide, H33258, Sigma-Aldrich; 1 μg/ml in PBS) were applied for counterstaining according to the manufacturers’ protocols.

Martin C., Gross V., Pflüger H.J., Stevenson P.A, & Mayer G. (2017). Assessing segmental versus non-segmental features in the ventral nervous system of onychophorans (velvet worms). BMC Evolutionary Biology, 17, 3.

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Immunofluorescence Staining of Tumor Tissue

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Receptor immunofluorescence staining was performed on dissected frozen tumor tissues. Tissues were fixed in 80% methanol for 5 min at 4 °C and acetone for 2 min at -20 °C. PBS was used for rehydration and sections were blocked with 12% bovine serum albumin/PBS for 30 min at room temperature. An EGFR-specific antibody (1:100; monoclonal mouse IgG, Dako) or a cMET-specific antibody (1:100; monoclonal mouse IgG1, R&D Systems) were used and sections were incubated for 1 h. Afterwards, sections were incubated with an anti-mouse Alexa488-conjugated secondary antibody (1:200; BD Pharmingen, Heidelberg, Germany). For nuclei counterstaining Hoechst bisbenzimide (1:1000; 5 mg/ml, Sigma-Aldrich) was applied and sections were embedded in Fluorescent Mounting Medium (Dako). Stained sections were examined using an Axiovert 135 TV fluorescence microscope equipped with an AxioCam MRm CCD camera and AxioVision Rel. 4.8 software (Carl Zeiss, Munich, Germany).

Urnauer S., Schmohl K.A., Tutter M., Schug C., Schwenk N., Morys S., Ziegler S., Bartenstein P., Clevert D.A., Wagner E, & Spitzweg C. (2019). Dual-targeted NIS polyplexes-a theranostic strategy toward tumors with heterogeneous receptor expression. Gene therapy, 26(3-4).

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