Nuclear dye hoechst 33342

Manufactured by Merck Group

Sourced in United States, Switzerland

Hoechst 33342 is a fluorescent dye that binds to the minor groove of DNA. It is commonly used in a variety of cell biology and biochemical applications as a nuclear stain.

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Lab products found in correlation

Lsm 710, by Zeiss (2 mentions) Rabbit anti th, by Abcam (1 mentions) D9663, by Merck Group (1 mentions) Fluoromount g mounting medium, by Southern Biotech (1 mentions) Vt1000, by Leica camera (1 mentions) Chicken anti tuj1, by Merck Group (1 mentions) Ethyl acetate, by Thermo Fisher Scientific (1 mentions) Methanol, by Thermo Fisher Scientific (1 mentions) Na2hpo4, by Carl Roth (1 mentions) O nitrophenyl β d galactopyranoside onpg, by Merck Group (1 mentions) Fk506, by Cayman Chemical (1 mentions) Potassium chloride (kcl), by ITW Reagents (1 mentions) Mncl2, by Merck Group (1 mentions) Nah2po4 h2o, by Merck Group (1 mentions) Milli q water purification system, by Merck Group (1 mentions) Mgso4.7h2o, by Merck Group (1 mentions) Dichloromethane, by Honeywell (1 mentions) Evos floid cell imaging station, by Thermo Fisher Scientific (1 mentions) A11004, by Thermo Fisher Scientific (1 mentions) A11001, by Thermo Fisher Scientific (1 mentions)

5 protocols using nuclear dye hoechst 33342

Identification of Activated Microglial Cells

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Microglial cells and/or peripheral monocytes were identified by immunocytochemistry with either anti-CD11b (Serotec; rat monoclonal antibody, clone 5C6; immunogen: thioglycollate-elicited peritoneal macrophages; dilution 1:100) or anti-CD45 (Serotec, rat monoclonal antibody, clone IBL-3/16; immunogen; purified B cells from mouse lymph nodes; dilution 1:40), as previously reported [31 (link)]. Both antibodies show equivalent labeling of microglial cells in organotypic cultures of the mouse retina [31 (link)]. Counterstaining was done with Hoechst 33342 nuclear dye (Sigma).
Some sections were double-immunolabeled with either anti-CD11b or anti-CD45 and anti-Poly-ADP ribosylated (PAR) polymers (Alexis Biochemicals, San Diego, CA, USA; mouse monoclonal antibody, clone 10H; immunogen: purified Poly-ADP ribose; dilution 1:50) to identify activated microglial cells. PAR polymers are produced by the activity of the enzyme Poly-ADP Ribose Polymerase-1 (PARP-1), which is increased in activated microglia [55 (link)].

Ferrer-Martín R.M., Martín-Oliva D., Sierra-Martín A., Carrasco M.C., Martín-Estebané M., Calvente R., Martín-Guerrero S.M., Marín-Teva J.L., Navascués J, & Cuadros M.A. (2015). Microglial Activation Promotes Cell Survival in Organotypic Cultures of Postnatal Mouse Retinal Explants. PLoS ONE, 10(8), e0135238.

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Immunostaining of Cultured hMOs

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Cultured and fixed hMOs were embedded in a 3% lowmelting-point agarose (Biozym) in PBS. Subsequently, 50 µm thick sections were cut using a vibratome (Leica VT1000s) and center-sections were used for assessing TH/FOXA2/TUJ1 expression. Prior to the immunostaining, sections were permeabilized using 0.5% Triton X-100 in PBS. Depending on the antibody, permeabilization times varied between 30 min and 2 h. Unspecific antigen blocking was achieved by incubating cut sections for 2 h in 2.5% donkey serum (Sigma-Aldrich, D9663), 2.5% BSA, 0.1% Triton X-100 and 0.1% sodium azide, followed by primary antibody incubation at 4 °C for 48 h on a shaker. Antibodies were diluted in blocking buffer as follows: rabbit anti-TH (1 : 1000, Abcam), chicken anti-TUJ1 (1 : 600, Millipore). This was followed by the incubation with secondary antibodies diluted in PBS containing 0.01% Triton X-100 and Hoechst-33342 nuclear dye (1 : 1000, Sigma-Aldrich). All secondary antibodies (Invitrogen) were conjugated to Alexa Fluor fluorochromes. Sections were mounted in Fluoromount-G mounting medium (Southern Biotech) and analyzed employing a confocal laser scanning microscope (Zeiss LSM 710).

Zanetti C ., Spitz S ., Berger E ., Bolognin S ., Smits LM ., Crepaz P ., Rothbauer M ., Rosser JM ., Marchetti-Deschmann M ., Schwamborn JC , & Ertl P . (2021). Monitoring the neurotransmitter release of human midbrain organoids using a redox cycling microsensor as a novel tool for personalized Parkinson's disease modelling and drug screening. The Analyst, 146(7).

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High-Pressure CO2 Extraction and Anti-Inflammatory Assay

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CO₂ pure grade (99.95%, Air Liquide, Lisbon, Portugal) and EtOH (96%) were used for high pressure extraction experiments. Solvents used for conventional extractions purification steps and chromatographic analysis included ethyl acetate (99.98%, Fisher scientific U.K. Limited, Loughborough, UK), methanol (99.8%, Fisher scientific U.K. Limited, Loughborough, UK), dichloromethane (Honeywell Riedel-de Haën, Germany), distilled water, and ultrapure water purified with a Milli-Q water purification system (Merck Millipore, Billerica, MA, USA). For the inflammation assays, the inflammatory stimulus was carried out by the usage of MnCl₂ (Merck, Darmstadt, Germany), and the anti-inflammatory positive control was carried by FK506 (Cayman Chemicals, Ann Arbor, MI, USA). Cellular lysis was preformed using Yeast Protein Extraction Reagent (Y-PER; ThermoFisher Scientific, Scientific, Rockford, IL, USA). To quantify the expression of reporter gene lacZ, we employed a solution buffer composed of Na₂HPO₄ (ROTH, Karlsruhe, Germany), NaH₂PO₄·H₂O (Merck, Buchs, Switzerland), KCl (Panreac, Barcelona, Spain), and MgSO₄·7H₂O (Merck, Buchs, Switzerland), containing o-nitrophenyl β-D-galactopyranoside (ONPG; Sigma–Aldrich^®–Poole, Dorset, UK). Nuclear staining was carried out using nuclear dye Hoechst 33342 (Sigma, Buchs, Switzerland).

Baixinho J.P., Anastácio J.D., Ivasiv V., Cankar K., Bosch D., Menezes R., de Roode M., dos Santos C.N., Matias A.A, & Fernández N. (2021). Supercritical CO2 Extraction as a Tool to Isolate Anti-Inflammatory Sesquiterpene Lactones from Cichorium intybus L. Roots. Molecules, 26(9), 2583.

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Immunofluorescence Analysis of Neurite Density

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The immunofluorescence was performed using anti-βIII tubulin antibody (Alexa 488-conjugated; Sigma-Aldrich) and with Nuclear dye Hoechst 33342 (0.25 µg/µL; Sigma-Aldrich). Randomly selected images were captured using an EVOS Floid Cell Imaging Station (Thermo Fisher Scientific Inc.) and analyzed with Nikon Imaging Software (NIS), the NIS- elements. The neurite density was assessed using the AutoQuant Neurite software (implemented in R program) and expressed as arbitrary units (AU) as described previously (Schonhofen et al., 2015 (link)).

Wollenhaupt-Aguiar B., Pfaffenseller B., Chagas V.D., Castro M.A., Passos I.C., Kauer-Sant’Anna M., Kapczinski F, & Klamt F. (2016). Reduced Neurite Density in Neuronal Cell Cultures Exposed to Serum of Patients with Bipolar Disorder. International Journal of Neuropsychopharmacology, 19(10), pyw051.

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Immunofluorescence Staining of Cellular Proteins

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Cells were seeded at 240,000 cells per well on autoclaved glass coverslips that were placed in 24-well plates. After treatment, cells were fixed with a mixture of 4% paraformaldehyde and 4% sucrose. The addition of 1% Triton X-100 to the fixative was used to determine the soluble from insoluble protein in the cell. After fixing, cells were permeabilized with 0.1% Triton X-100 and then blocked with 3% BSA in PBS. Cells were probed with antibodies for p-Ser-129 using either Affinity Bioreagents (PA1-4686 1:2000) for Fig. 1G or Covance-81A (MMS-5091 1:5000) for the remaining figures. For non-αSyn staining, cells were plated and fixed as above but were blocked with 10% horse serum and 5% FBS in PBS. Cells were probed with either LC3 (L8918 Sigma 1:500), MAP2 (Sigma M4403 1:1000), or GFAP (Dako Z0334 1:500). Alexa Fluor 488 (Invitrogen A11001, A11008 1:500) or 568 (Invitrogen A11004 1:500) secondary antibodies were subsequently added to the wells. Cells were then counter stained with nuclear dye Hoechst 33342 (Sigma 861405) and mounted with Fluoromount-G (Southern Biotechnology). All images were acquired using a Leica TCS SP5 V confocal laser scanning microscope.

Redmann M., Wani W.Y., Volpicelli-Daley L., Darley-Usmar V, & Zhang J. (2017). Trehalose does not improve neuronal survival on exposure to alpha-synuclein pre-formed fibrils. Redox Biology, 11, 429-437.

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