Hoechst nuclear stain

Manufactured by Thermo Fisher Scientific

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Hoechst nuclear stain is a fluorescent dye used to stain the nuclei of cells. It binds to DNA and emits blue fluorescence when excited by ultraviolet or violet light.

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Lab products found in correlation

50 protocols using hoechst nuclear stain

Brain Tissue Preparation and Imaging

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For ReNL reporter analysis, 6 days after injection, mice were anesthetized and perfused with 4% paraformaldehyde. Brains were extracted, postfixed overnight, and soaked in 30% sucrose for 24 hours. Brains were frozen on dry ice and mounted onto a cryostat sample holder using Optimal Cutting Temperature compound, cryosectioned (coronal plane sections) using a Leica CM 3050 S cryostat (Leica Biosystems), and the prepared 40-μm sections were mounted onto glass slides with Hoechst nuclear stain (1:4000 dilution) and SlowFade Gold Antifade Reagent (Thermo Fisher Scientific). Mounted sections were stored at −80°C and protected from light until use. Sections were imaged by fluorescence microscopy using a Zeiss Apotome.2 microscope with Zen Blue software. Microscope settings were maintained across all image acquisition.

Rui Y., Wilson D.R., Choi J., Varanasi M., Sanders K., Karlsson J., Lim M, & Green J.J. (2019). Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing. Science Advances, 5(12), eaay3255.

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Multimarker Immunofluorescence Staining

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Sections were washed with PBS for 5 min, then permeabilized and blocked in a PBS solution containing 1% BSA, 0.3% Triton X-100, 2% (v/v) donkey serum, and 0.02% sodium azide for 30 min at room temperature. Sections were incubated with primary antibodies in blocking solution overnight at 4°C. Primary antibodies include anti-MCHR1 (rabbit pAB; 1:250 dilution; catalog #711649, Thermo Fisher Scientific), anti-adenylate cyclase 3 [ACIII; 1:1000 dilution; chicken polyclonal antibody (pAb); CPCA-ACIII, Encor], anti-mCherry (chicken pAb; 1:1000 dilution; catalog NBP2-25158, Novus), anti-MCH (1:200 dilution; rabbit mAb; catalog #274415, Abcam). Sections were then washed with PBS before incubating with secondary antibodies for 1 h at room temperature. Secondary antibodies include donkey conjugated Alexa Fluor 647 and 488 (1:1000; Thermo Fisher Scientific) against appropriate species according to the corresponding primary. All primary and secondary solutions were made in the blocking solution described above. Slides were then washed in PBS and stained with Hoechst nuclear stain (catalog #H3570, Thermo Fisher Scientific) for 5 min at room temperature. Coverslips were mounted using SlowFade Diamond Antifade Mountant (catalog #S36972, Thermo Fisher Scientific).

Brewer K.M., Engle S.E., Bansal R., Brewer K.K., Jasso K.R., McIntyre J.C., Vaisse C., Reiter J.F, & Berbari N.F. (2023). Physiological Condition-Dependent Changes in Ciliary GPCR Localization in the Brain. eNeuro, 10(3), ENEURO.0360-22.2023.

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Immunostaining of Decapitated C. elegans

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Adult C. elegans were washed in M9 buffer and decapitated with a syringe needle to expel the intestine. Tissues were fixed overnight at 4°C in 4% formalin (Sigma-Aldrich HT5011) then blocked for 1 hour at room temperature in PBS+ (1x PBS with 0.1% Triton X-100, 1% bovine serum albumin, 1% donkey serum, and 0.02% sodium azide). Rabbit anti-FLAG primary antibody (Thermo Fisher Scientific, Cat. No. 740001; 1:1,000) and mouse anti-HDEL primary antibody (previously used by (32 (link)) to mark the ER in C. elegans; Santa Cruz Biotechnology, Santa Cruz, CA; 1:250) were diluted in PBS+ and tissues were incubated occurred overnight at 4°C. Secondary antibody incubation with Alexa Fluor 555 goat anti-rabbit IgG (Thermo Fisher Cat no. A21428; 1:8,000 dilution) was for 1 hour at room temperature. Tissues were washed three times for 5 minutes each with PBS with 0.1% Triton X-100 in between antibody incubation periods. The final wash contained Hoechst nuclear stain (Thermo Fisher 33258) at 1:1,000 dilution to visualize nuclei and phalloidin stain (Invitrogen Alexa Fluor 488 Phalloidin) at 1:300. Confocal images were taken with a Nikon Ti2 spinning disk confocal with a Yokohama X1 disk and an Orca Flash4.0 sCMOS (Hamamatsu). Images were acquired in Nikon Elements AR 5.0.

Zein-Sabatto H., Cole T., Hoang H.D., Tiwary E., Chang C, & Miller M.A. (2020). The type II integral ER membrane protein VAP-B homolog in C. elegans is cleaved to release the N-terminal MSP domain to signal non-cell-autonomously. Developmental biology, 470, 10-20.

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Mitochondrial Protein Localization Assay

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Mouse monoclonal anti-mitofusin 2 was from AbCAM (Cat#: ab56889; 1:1000 dilution), rabbit polyclonal anti-cytochrome c oxidase subunit 4 (COX-IV) was from AbCAM (Cat#: ab16056; 1:1000 dilution), mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase was from AbCAM (Cat#: ab8245; 1:3000 dilution), α-bungarotoxin Alexa-Fluor 594 was from ThermoFisher (Cat#: B12423; 0.5 μg/ml), Alexa-Fluor 488 goat anti-rabbit was from ThermoFisher (Cat#: A11008; 1:400 dilution), fluorescein-conjugated wheat germ agglutinin was from Invitrogen (Cat#: W834; _1:50 dilution), MitoTracker Orange was from Thermo Fisher (Cat#: M7510), tetramethylrhodamine ethyl ester was from Thermo Fisher (Cat#: T669), and Hoechst nuclear stain was from Thermo Fisher (Cat#: H3570).

Franco A., Dang X., Zhang L., Molinoff P.B, & Dorn GW I.I. (2022). Mitochondrial Dysfunction and Pharmacodynamics of Mitofusin Activation in Murine Charcot-Marie-Tooth Disease Type 2A. The Journal of Pharmacology and Experimental Therapeutics, 383(2), 137-148.

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Quantifying SPIO Nanoparticle Uptake

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The uptake was studied as described previously.26 (link) Briefly, cells were incubated with SPIO or SPIO-Serum for 1 h or 4 h, washed with PBS to remove the free nanoworms and passed over a Mini MACS magnetic column (Miltenyi Biotec) to separate IRON labeled from unlabeled cells. Magnetic cells from different groups were suspended in equal amounts of 1% (wt/vol) BSA-PBS and concentrated on glass slides using cytospin (Thermo Fisher Scientific, Rockford, IL, USA). The cells on slides were then fixed with 10% buffered formalin solution and stained with Hoechst nuclear stain (Thermo Fisher Scientific, Rockford, IL, USA). Random microscopic areas were used for cell counting.

Zhang Y., Xia M., Zhou Z., Hu X., Wang J., Zhang M., Li Y., Sun L., Chen F, & Yu H. (2021). p53 Promoted Ferroptosis in Ovarian Cancer Cells Treated with Human Serum Incubated-Superparamagnetic Iron Oxides. International Journal of Nanomedicine, 16, 283-296.

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Lysosome Staining of Gene-Edited Cells

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Gene-edited cells were seeded in 24 well plates at a density of 5 × 10⁵ cells per well for 24 h. Lysotracker (Beyotime, C1046) was diluted in DMEM medium at a ratio of 1:1000. After staining for 10 min, the Hoechst nuclear stain (Thermo) was added to the medium at a ratio of 1:500. After incubating for 10 min at 37 °C, the cells were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min. The fluorescence intensity was observed using a confocal microscope, and representative cells were selected and photographed.

Wang Y., Peng Y., Zi G., Chen J, & Peng B. (2024). Co-delivery of Cas9 mRNA and guide RNAs for editing of LGMN gene represses breast cancer cell metastasis. Scientific Reports, 14, 8095.

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Fluorescent Imaging of Cell Populations

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LNCaP and PC-3 cells were trypsinized, counted, and resuspended to a final concentration of

1.5 \times 10^{5}

cells per well into a 96 well glass bottom plate (Cellvis P96-1.5H-N, Mountain View, California, United States). Cells were fixed with 4% PFA for 15 min at RT, followed by two washes with 1x PBS for 5 min each at RT. Cells were then blocked with 5% FBS in 1x PBS for 15 min at RT. Blocking solution was then carefully removed without additional washing. The cells were then stained with the J533-AF647 conjugate for 30 min at RT with final staining concentrations of 1, 5, 10, and

20 μ g / mL

. Cells were washed with 1x PBS + 0.5% Triton-X for 5 min at RT, followed by a wash with 1x PBS for 5 min at RT. Cell staining with the J533-AF647 conjugate was compared to an unstained control sample. A Hoechst nuclear stain (Thermo Fisher Scientific) was performed on all samples with a final staining concentration of

1 μ g / mL

. The cells were then imaged at

20 \times

on a Yokogawa CSU-X1 Zeiss Axio Observer microscope (Oberkochen, Germany) in the UV and Cy5 channels.

Kwon M.J., House B.J., Barth C.W., Solanki A., Jones J.A., Davis S.C, & Gibbs S.L. (2023). Dual probe difference specimen imaging for prostate cancer margin assessment. Journal of Biomedical Optics, 28(8), 082806.

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Immunostaining of Brain Sections

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Brain sections were treated overnight with primary antibody in 5% normal donkey serum/PBS with 0.1% Tween-20 at 4 °C and followed by the appropriate fluorescently conjugated secondary antibody. For samples requiring further unmasking of the epitope, which included anti-Aldh1L1, antigen retrieval was performed by boiling in 6 M sodium citrate buffer at pH 6.0 for 20 min. Slides were then cooled to room temperature and washed 3× with PBS and 0.1% Tween-20, and the standard immunostaining protocol was followed. Well characterized primary antibodies were as follows: rabbit anti-Iba1 (1:500, WAKO), goat anti-Iba1 (Abcam; 1:300), rat anti-SF1 (1:800, kindly provided by Dr. Taro Tachibana, Osaka City University JAPAN), mouse anti-Olig2 (1:300, Millipore), rat anti-LAMP1 (1:800, Millipore), rat anti-CD68 (1:500, BIORAD), goat anti-Sox9 (1:60, R&D systems), rabbit anti-NKX2.1 (1:400, Santa Cruz), sheep anti-Csf1R (1:300, R&D Systems), goat anti-PdgfR alpha (1:150, R&D Systems), rabbit anti-GFAP (1:500; DAKO), rabbit anti-SB100 (1:400; DAKO), and rabbit anti-Aldh1l1 (1:500, Abcam). All appropriate secondary antibodies were Donkey anti-IgG Alexa Fluor conjugated (1:400, ThermoFisher Scientific). All samples were counterstained with Hoechst nuclear stain (1:1000; ThermoFisher Scientific H3570).

Marsters C.M., Nesan D., Far R., Klenin N., Pittman Q.J, & Kurrasch D.M. (2020). Embryonic microglia influence developing hypothalamic glial populations. Journal of Neuroinflammation, 17, 146.

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Embryonic Patterning and Staining Protocols

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Embryos were dissected in cold phosphate-buffered saline and processed for β-galactosidase activity or immunofluorescence as previously described [50 (link)]. Antibodies used were: Shh, Nkx2.2 (Developmental Studies Hybridoma Bank; 1:5); FoxA2 (Cell Signaling; 1:500); Olig2 (Millipore; 1:300); Arl13b (NeuroMab 455-8JD-29; 1:500); Smo (kindly provided by K Anderson; 1:500); Alexa Fluor 488 and Alexa Fluor 568 (1:300, ThermoFisher); and Hoechst nuclear stain (1:3000). Alizarin red and alcian blue staining were performed as previously described [51 (link), 52 (link)].

Gigante E.D., Long A.B., Ben-Ami J, & Caspary T. (2018). Hypomorphic Smo mutant with inefficient ciliary enrichment disrupts the highest level of vertebrate Hedgehog response. Developmental biology, 437(2), 152-162.

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Multimodal Cell Culture Assay

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RPMI, neurobasal medium, FBS, L-glutamine, IR dye–tagged secondary antibodies, Hoechst nuclear stain, penicillin, streptomycin, horse serum, and other cell culture reagents were purchased from Thermo Fisher Scientific. The Bradford protein assay kit was purchased from MilliporeSigma. The Phalloidin antibody and protease and phosphatase inhibitor cocktail were ordered from Thermo Fisher Scientific, acetylation inhibitor cocktail from Santa Cruz Biotechnology, and propidium iodide (PI) from Molecular Probes.

Huang M., Lou D., Charli A., Kong D., Jin H., Zenitsky G., Anantharam V., Kanthasamy A., Wang Z, & Kanthasamy A.G. (2021). Mitochondrial dysfunction–induced H3K27 hyperacetylation perturbs enhancers in Parkinson’s disease. JCI Insight, 6(17), e138088.

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