Hoechst staining solution

Manufactured by Thermo Fisher Scientific

Hoechst staining solution is a fluorescent dye used for the detection and visualization of DNA in biological samples. It binds to the minor groove of DNA, emitting a blue fluorescent signal when excited by ultraviolet light. This solution is commonly used in various applications, such as cell biology, molecular biology, and microscopy, to stain and identify nucleic acids.

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Lab products found in correlation

Vt100, by Leica (2 mentions) Fluoromount, by Merck Group (2 mentions) Eclipse 90i epifluorescence microscope, by Nikon (2 mentions) Lsm 700 confocal laser scanning microscope, by Zeiss (2 mentions) Mitosox, by Thermo Fisher Scientific (2 mentions) Tcs nt, by Leica (1 mentions) Dcfh da, by Beyotime (1 mentions)

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Hoechst (1591 citations) Hoechst solution (53 citations) Hoechst staining (32 citations) Hoechst 33342 staining solution (12 citations)

4 protocols using hoechst staining solution

Immunohistochemistry Protocol for Cortical Sections

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80 μm coronal sections were performed using a vibratome (Leica, #VT100S). Sections were permeabilized in PBST (0.3% Triton X-100, diluted in PBS 1X) and incubated for two hrs at room temperature in blocking solution (10% Horse Serum Albumine in PBST), then overnight at 4°C with primary antibodies. Treatment with HCl 2N at 37°C for 30′ was performed before incubation with standard blocking solution for KI67 immunohistochemistry. Treatment with Na citrate pH 6 at 80°C for 40′ was performed before incubation with standard blocking solution for RORB immunohistochemistry.
Sections were rinsed three times in PBST and incubated for 2 hrs at room temperature with corresponding secondary antibodies (1:500, Life Technologies). Three washes in PBST were performed, followed by 10 min incubation with Hoechst staining solution (1:5000 in PBS 1X, Life Technologies) to label nuclei, before dry mounting on slides with Fluoromount (Sigma). For imaging, the putative primary somatosensory cortex (S1) was used as region of study for all the experiments. Images were acquired on Eclipse 90i epifluorescence microscope (Nikon) or on LSM 700 confocal laser scanning microscope (Carl Zeiss).

Tomasello U., Klingler E., Niquille M., Mule N., Santinha A.J., de Vevey L., Prados J., Platt R.J., Borrell V., Jabaudon D, & Dayer A. (2022). miR-137 and miR-122, two outer subventricular zone non-coding RNAs, regulate basal progenitor expansion and neuronal differentiation. Cell Reports, 38(7), 110381.

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Immunohistochemical Analysis of Cortical Development

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Embryos were collected 24 h or 48 h following in utero electroporation and post-fixed overnight in 4% paraformaldehyde (PFA, Sigma) at 4°C. Postnat al mice from P0 were perfused with 4% PFA and post-fixed overnight in 4% PFA at 4°C. Fift y to 70 µm coronal sections were performed using a vibrating microtome (Leica, #VT100S). Immunofluorescence staining was performed as followed: sections were incubated for one hour at room temperature in blocking solution (3% Bovine Serum Albumine, 0,3% Triton X-100, diluted in PBS 1X), then overnight at 4°C with primary antibodies. Treatment with HCl 2 N at 37°C for 40’ was performed after incubation with standard blocking solution for BrdU immunohistochemistry. Sections were rinsed three times in PBS 1X and incubated for 1 hour at room temperature with corresponding secondary antibodies (1:500, Life Technologies). Three washes in PBS 1X were performed, the second one using Hoechst staining solution (1:10000 in PBS 1X, Life Technologies) to label nuclei, before dry mounting on slides with Fluoromount (Sigma). For imaging, the putative primary somatosensory cortex (S1) was used as region of study for all the experiments. Images were acquired on Eclipse 90i epifluorescence microscope (Nikon) or on LSM 700 confocal laser scanning microscope (Carl Zeiss).

Vitali I., Fièvre S., Telley L., Oberst P., Bariselli S., Frangeul L., Baumann N., McMahon J.J., Klingler E., Bocchi R., Kiss J.Z., Bellone C., Silver D.L, & Jabaudon D. (2018). Progenitor hyperpolarization regulates the sequential generation of neuronal subtypes in the developing neocortex. Cell, 174(5), 1264-1276.e15.

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Quantifying Cellular ROS and Mitochondrial Superoxide

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A broad spectrum of ROS were detected in living cells using the DCFH-DA (Beyotime). Dilute the 10 mM DCFH-DA in medium without FBS to make a 10 μM DCFH-DA working solution. Apply 2 mL of 10uM DCFH-DA to cover cells for 20mins at 37 °C, protected from light. Wash cells gently three times with phophate buffered saline (PBS). Mitochondrial superoxide was detected in living cells using the fluorogenic dye MitoSOX (invitrogen). Dissovle the 50μg MitoSOX component in 13uL of Dimethyl sulfoxide (DMSO) to make a 5 mM MitoSOX reagent stock solution. Dilute the 5 mM MitoSOX reagent stock solution in medium without FBS to make a 5 μM MitoSOX reagent working solution. Apply 2 mL of 5uM MitoSOX reagent working solution to cover cells for 10mins at 37 °C, protected from light. Wash cells gently three times with PBS. Then the cells were covered with 1μg/ml Hoechst staining solution (Thermofisher). The cells were observed by using a confocal laser scanning microscope (Leica TCS-NT, Germany). The acquired images were converted into binary images for the quantification of the average fluorescence intensity using ImageJ software (National Institutes of Health, USA).

Liang X., Wang Z., Gao M., Wu S., Zhang J., Liu Q., Yu Y., Wang J, & Liu W. (2019). Cyclic stretch induced oxidative stress by mitochondrial and NADPH oxidase in retinal pigment epithelial cells. BMC Ophthalmology, 19, 79.

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Intracellular ROS Detection in Live Cells

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Intracellular reactive oxygen species (ROS) generation was detected in living cells by a DCF-DA kit, and mitochondrial superoxide was detected by fluorogenic dye MitoSOX (Invitrogen). We seeded cells in an 8-well chamber slide at a density of 5 × 10⁴ cells/mL and incubated them for 24 h at 37°C. We washed them three times with PBS and added 10 μM of chloromethyl 2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) for 20 min or 5 μM MitoSOX for 10 min to the cells in the dark. After incubation, we washed the cells gently three times with PBS and added 1 μg/ml Hoechst staining solution (Thermofisher) to them. These cells were observed by use of fluorescence microscopy. The acquired images were converted into binary images for the quantification of the average fluorescence intensity using ImageJ software. We set the related fluorescence unit of the control group as 1. We repeated each experiment three times.

Lin H.D., Lee Y.C., Chiang C.Y., Lin Y.J., Shih C.Y., Tsai R.K., Lin P.Y., Lin S.Z., Ho T.J, & Huang C.Y. (2023). Protective effects of Scoparia dulcis L. extract on high glucose-induced injury in human retinal pigment epithelial cells. Frontiers in Nutrition, 10, 1085248.

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