Hoechst 33258 fluorochrome

Manufactured by Merck Group

Hoechst 33258 is a fluorochrome compound commonly used in fluorescence microscopy. It is a blue-fluorescent dye that specifically binds to the minor groove of DNA, and its fluorescence is enhanced upon binding to DNA.

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Lab products found in correlation

Bx51 microscope, by Olympus (2 mentions) Diaminobenzidine dab kit, by Thermo Fisher Scientific (1 mentions) Tracp kit, by Merck Group (1 mentions) Confocal laser scanning microscope, by Leica (1 mentions)

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Hoechst 33258 (1476 citations) Hoechst (711 citations)

4 protocols using hoechst 33258 fluorochrome

Osteoclast Differentiation and Bone Resorption

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To evaluate the differentiation of hematopoietic precursor cells to osteoclasts, bone slices having osteoclasts were stained with a TRACP-kit (Sigma) at +37 °C according to the manufacturer’s instructions. The nuclei were stained with the DNA-binding Hoechst 33258 fluorochrome (Sigma) for 10 min at room temperature. TRACP-positive cells having three or more nuclei were considered as osteoclasts and were counted from bone slices using a fluorescence microscope (Zeiss AX10; Carl Zeiss Ltd., Hertfordshire, England) with a 20×/NA 0.5 objective (Zeiss).
After counting the osteoclasts, the cells were removed from the bone slice by gentle scraping and the slices were dyed with peroxidase-conjugated WGA-lectin (WGA; 20 µg/ml) for 30 min at room temperature. Slices were washed 3 times with PBS and counterstained using a diaminobenzidine (DAB) kit according to the manufacturer’s instructions (Invitrogen, CA, USA). The total area of resorption pits was measured by acquiring images of each bone slice with a fluorescence microscope (Zeiss AX10), 10×/NA 0.25 Objective (Zeiss) and QImaging Retiga 4000R camera with a digital image analyzer (MCID Core 7.0, Ontario, Canada). Ranges of interest (ROIs) were drawn for each resorption pit and analyzed with FIJI (ImageJ) software^{40 (link)}.

Koskela A., Koponen J., Lehenkari P., Viluksela M., Korkalainen M, & Tuukkanen J. (2017). Perfluoroalkyl substances in human bone: concentrations in bones and effects on bone cell differentiation. Scientific Reports, 7, 6841.

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TUNEL Assay for Apoptosis Detection

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Lung tissue sections were fixed in 4% paraformaldehyde, rinsed with PBS solution, incubated with 0.5% TritonX-100 for 20 min. Endogenous peroxidase activity was blocked using 3% H₂O₂ in 10 mM Tris. The treated tissues were placed in equilibration buffer and incubated with TdT enzyme in a humid chamber at 37°C for 2 hrs. The reaction was terminated with stop buffer and incubated with a biotin-conjugated rat anti-digoxin antibody and SABC-FITC (1:100; Boster Bio-Engineering Limited Company, China) for 30 min. respectively. Hoechst 33258 fluorochrome (Sigma-Aldrich) was used for nuclear staining. After washing with PBS, tissue sections were mounted in neutral glycerine and analysed under a laser scanning confocal microscope (Leica, Wetzlar, Germany). The fluorescence imaging was conducted with excitation at 488 nm and emission at 510 nm at room temperature.

Song X., Wang B., Lin S., Jing L., Mao C., Xu P., Lv C., Liu W, & Zuo J. (2014). Astaxanthin inhibits apoptosis in alveolar epithelial cells type II in vivo and in vitro through the ROS-dependent mitochondrial signalling pathway. Journal of Cellular and Molecular Medicine, 18(11), 2198-2212.

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Immunofluorescence Staining of Glial Cells

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Cells grown on uncoated glass coverslips were used for indirect and double immunofl uorescence staining. The intermediate fi lament proteins (GFAP, vimentin, neurofi laments), MAP2, ferritin, fi bronectin and F VIII were detected on cells fi xed in methanolacetone (1:1) solution for 15 min. at (-15 °C). The surface antigen CD11c was examined on cells fi xed in 4 % paraformaldehyde for 15 min at room temperature.
Cells for indirect immunofl uorescence were incubated 1 h with primary antibodies and 30 min with 1:50 diluted appropriate secondary antibodies. Double-labelling was performed with primary, and afterwards with appropriate mixtures of secondary antibodies for 1 h and 30 min, respectively. Nuclei were stained with Hoechst 33258 fl uorochrome (5 μg/ml in PBS, Sigma) for 1 min. To determine the percentage of immunoreactive glial cell types, 20 fi elds were enumerated at 200x magnifi cation on confl uent cultures.
Staining of brain cryosections (10 um thick) was performed by the same immunofl uorescence methods as described above. Formalin-fi xed, paraffi n-embedded sections were stained for ferritin, 1 h incubation with peroxidase-conjugated antirabbit secondary antibodies (Dako). The diaminobenzidine reagent was used as the chromogen. Fluorescence microscopy was performed using an Olympus BX51 microscope (Hamburg, Germany).

Sivakova I., El Falougy H., Kubikova E, & Perzelova A. (2022). Uncertain histological origin of "glia-like" cells in adult human brain tissue cultures. Bratislavske lekarske listy, 123(9).

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Immunofluorescence Analysis of Cell Cultures

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Indirect immunofl uorescence using antibodies to IF was performed in all 28 cultures. Cells grown on coverslips were rinsed with PBS, fi xed in methanol-acetone (1:1) solution for 15 min at -15 °C. We used the following antibodies: against GFAP, clone GF-01, 1:100 (Exbio, Prague), and polyclonal sera to GFAP, 1:100 (Dako); to vimentin clone V9, 1:100 (Sigma, Germany); to cytokeratins monoclonal anti-pan CK types: 1,4,5,6,8,10,13,18,19 (Sigma); to neurofi laments clone NF-01, 1:100 (Exbio); to nestin clone 25, 1:50 (BD Biosciences, USA) and polyclonal sera to nestin 1:100 (Millipore, USA). Secondary fl uorescein-conjugated antibodies were purchased from Sigma and Sevapharma (Prague). Cells for indirect immunofl uorescence were incubated 1h with primary and 30 min. with secondary antibodies. Nuclei were stained with Hoechst 33258 fl uorochrome (5 μg/ml in PBS, Sigma) for 1 min. To determine the percentage of immunoreactive cells, 30 fi elds were enumerated at 200x magnifi cation, equally distributed over the coverslips fi xed at different DIV and passage numbers. Fluorescence micoscopy was performed using an Olympus BX51 microscope (Olympus, Germany).

Sivakova I., MacLeod R., Mraz P., Kubikova E, & Perzelova A. (2019). Short-term glioblastoma cultures may contain normal "glia-like" cells. Bratislavske lekarske listy, 120(9).

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