H1399

Manufactured by Thermo Fisher Scientific

Sourced in United States

The H1399 is a laboratory equipment product from Thermo Fisher Scientific. It is a high-precision device designed for specific laboratory applications. The core function of the H1399 is to perform measurements and analysis tasks within controlled laboratory environments.

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Lab products found in correlation

Hoechst 33342, by Thermo Fisher Scientific (14 mentions) Dmi8 fluorescent microscope, by Leica (3 mentions) Zen blue software, by Zeiss (2 mentions) A21207, by Thermo Fisher Scientific (2 mentions) Goat anti mouse igg h l dylight 594, by Bioworld Technology (2 mentions) Bs10027, by Bioworld Technology (2 mentions) Lsm 710, by Zeiss (2 mentions) Elements viewer, by Nikon (1 mentions) F3165, by Merck Group (1 mentions) Ab27985, by Abcam (1 mentions) Ab16287, by Abcam (1 mentions) A11055, by Thermo Fisher Scientific (1 mentions) Ab131259, by Abcam (1 mentions) Ab33168, by Abcam (1 mentions) Ab216327, by Abcam (1 mentions) Phdr1, by Cytoskeleton (1 mentions) Tcs sp8, by Leica (1 mentions) Mitosox red reagent, by Thermo Fisher Scientific (1 mentions) Millicell ez chamber slides, by Merck Group (1 mentions) Prism 8, by GraphPad (1 mentions)

Close spelling variants of this product

Hoechst 33342 (H1399) (18 citations) Hoechst (H1399; (5 citations) Hoechst 43222 (H1399) (2 citations)

31 protocols using h1399

Photomanipulation of Cellular Dynamics

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Cells were captured in the mCherry channel only, to visualize the sspB component before activation. Cells were then activated with the 488 nm laser (typically for 3 min) with a frame interval of 2 s, while imaging in GFP and mCherry at Nyquist zoom. For local activation, a 1 µm² square is activated. For following compaction over time, the 3 min activation was followed by 30 min of 5 s intervals of activation.
For offline activation, cells were placed on an LED array (Amuza). After activation for an indicated amount of time, 4% formaldehyde is added to the wells. After 10 min incubation, cells are washed with phosphate-buffered saline (PBS; Thermo Fisher 14190250). The cells are then treated with 1:2000 Hoechst (Thermo Fisher H1399) in PBS for 20 min or continued to IF.

Eeftens J.M., Kapoor M., Michieletto D, & Brangwynne C.P. (2021). Polycomb condensates can promote epigenetic marks but are not required for sustained chromatin compaction. Nature Communications, 12, 5888.

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Immunofluorescence Staining of Frozen Tissue Sections

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The frozen tissues were mounted on the specimen stages in the same way as described for “in situ hybridization”. Sections with 12 µm thickness were mounted onto a poly-l-lysine coated slide. Sections were fixed in cold 4% paraformaldehyde for 10 min after air drying. Slides were then processed for IF staining. IF was performed as previously described^{2 (link),4 (link)}. Frozen sections (12 µm) from each genotype were processed onto the same slides and incubated with primary antibodies listed in Supplementary Table 2. For signal detection, secondary antibodies listed in Supplementary Table 2 were used. Nuclear staining was performed using Hoechst 33342 (5 µg/mL, H1399, Thermo Scientific). Pictures were taken using the Nikon Eclipse 90i upright microscope and processed by Nikon Elements Viewer.

Yuan J., Aikawa S., Deng W., Bartos A., Walz G., Grahammer F., Huber T.B., Sun X, & Dey S.K. (2019). Primary decidual zone formation requires Scribble for pregnancy success in mice. Nature Communications, 10, 5425.

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Immunofluorescence Analysis of Transfected Cells

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The AN3 CA cells were seeded on coverslips (Marienfeld-Superior). After 48 h, cells were transfected. After another 48 h, transfected cells were fixed with 3.7% paraformaldehyde for 15 min followed by permeabilization with 0.1% Triton X-100 for another 15 min. The cells were blocked with 5% goat serum at 25° C for 1 h, followed by the incubation with primary antibody at 4° C for 16 h. The cells were then washed 3 times with 1X PBS for 5 min each. The secondary antibody was used to incubate cells at 25° C for 1 h. The cells were then washed 5 times with 1X PBS for 5 min each. The primary and secondary antibodies used were anti-flag (1:200; F3165, Sigma-Aldrich) and goat anti-mouse IgG (H+L) Cy3 conjugate (1:400; 81-6515, Zymed, Thermo Fisher Scientific). The cell nuclei were stained with Hoechst 33342 (1:400; H-1399, Thermo Fisher Scientific) at 25° C for 5 min. Cell images were acquired using a confocal microscope Zeiss LSM (Zeiss) and images were analyzed using Fiji software (Version 2.0.0-rc-69/1.52n, NIH).

Chen Z.S., Lin X., Chan T.F, & Chan H.Y. (2021). Pan-cancer investigation reveals mechanistic insights of planar cell polarity gene Fuz in carcinogenesis. Aging (Albany NY), 13(5), 7259-7283.

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Pluripotency Marker Expression in hiPSCs

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Human iPSCs were fixed in 4% PFA at room temperature and then stained with antibodies against the following makers: Oct3/4 (1:500 dilution Abcam #ab27985) and SSEA-4 (1:100 dilution Abcam# ab16287) and AlexFluor-488 conjugated antibodies against human TRA-1-60 (1:100 dilution BD Pharmigen #560173) in 0.1% Triton (Fisher X-100) and and 1% Fetal Bovine Serum (FBS) in PBS. For secondary antibodies goat IgG (1:250 in PBS, Invitrogen #A11055) and mouse IgG (1:250 in PBS, Invitrogen #A11055) were used to detect Oct3/4 and anti-SSEA-4, respectively. Cell nuclei were counterstained with Hoechst 33342 (10 ng/mL of Thermo Scientific #H1399).

Luzzi A., Wang F., Li S., Iacovino M, & Chou T.F. (2023). Skeletal muscle cell protein dysregulation highlights the pathogenesis mechanism of myopathy-associated p97/VCP R155H mutations. Frontiers in Neurology, 14, 1211635.

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Immunostaining of Endothelial Tight Junctions

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HBMECs grown on collagen-coated coverslips in 24-well culture plates were washed in PBS and ﬁxed in 4% paraformaldehyde for 15 min at room temperature. Fixed cells were blocked with 5% BSA and permeabilized with 0.1% Triton X-100 for 30 min at room temperature. HBMECs were then immunostained with VE-cadherin (1:200, ab33168, Abcam), claudin-5 (1:100, ab131259, Abcam), ZO-1 (1:50, 40–2200, Thermo Fisher) or occludin (1:100, ab216327, Abcam) diluted in blocking buffer overnight at 4 °C and washed three times with PBS at 5-min intervals. F-actin staining was performed by using Rhodamine-Phalloidin (100 nmol/L, PHDR1, Cytoskeleton, Denver, CO, USA) at room temperature for 1 h. HBMECs were then incubated with Alexa488-anti-rabbit or Alexa488-anti-mouse secondary antibodies (Molecular Probes, Carlsbad, CA, USA) for 1 h followed by counterstaining the nuclear with Hoechst 33342 (1:10000, H1399, Thermo Fisher) for 15 min at room temperature. Images were captured by confocal microscopy using a Leica TCS SP8 (Leica Microsystems, Buffalo Grove, IL, USA).

Tang J., Kang Y., Huang L., Wu L, & Peng Y. (2020). TIMP1 preserves the blood–brain barrier through interacting with CD63/integrin β1 complex and regulating downstream FAK/RhoA signaling. Acta Pharmaceutica Sinica. B, 10(6), 987-1003.

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Mitochondrial Superoxide Detection Using MitoSOX

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MitoSOX Red reagent (Thermo Fisher Scientific, M36008) was used to detect mitochondrial superoxide levels in live cells. MG‐63 cells were cultured in Millicell EZ chamber slides (EMD Millipore). A 5 mM MitoSOX Red reagent stock solution was made by dilution into dimethyl sulfoxide (DMSO). A 5 μM MitoSOX Red reagent working solution was made by diluting the stock into a culture medium. Cells were loaded with MitoSOX Red reagent by incubating for 10 minutes at 37°C protected from light. Hoechst 33342 (1:2000) live‐cell dye was used as a counterstain to detect the nuclei of live cells (Thermo Fisher Scientific, H1399). Cells were washed three times with a warm medium. Intensity measurements were obtained using the Zen Blue software (Zeiss) analyzed using Prism 8 (GraphPad). Rotenone (Sigma, St. Louis, MO, USA; R8875) was applied as a positive control. For each replicate (n = 6 replicates/condition), average ratios were derived from four different fields of views of 5 to 10 individual cells. Data are presented as mean ± SEM error bars; ^****p ≤ 0.0001, ^***p ≤ 0.001, ^**p ≤ 0.01, and ^*p ≤ 0.05 (one‐way ANOVA with Tukey's multiple comparisons test compared with vehicle).

Quigley M., Rieger S., Capobianco E., Wang Z., Zhao H., Hewison M, & Lisse T.S. (2021). Vitamin D Modulation of Mitochondrial Oxidative Metabolism and mTOR Enforces Stress Adaptations and Anticancer Responses. JBMR Plus, 6(1), e10572.

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Immunocytochemistry of iPSC-derived Motor Neurons

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For ICC performed on iPSC-derived spinal motor neurons, neurons were fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.2% Triton X-100 for another 15 min. The cells were then blocked with 5% donkey serum at 25 °C for 1 h before being incubated with primary antibodies. The primary antibodies used were anti-PITX2 (1:200, H00005308-M01, Novus Biologicals), anti-Tau (1:500, ab75714, Abcam), anti-Bassoon (1:200, ab110426, Abcam), anti-MAP2 (1:1000, ab5392, Abcam), and anti-Homer1 (1:200, ab97593, Abcam). Secondary antibodies used in this study were Alexa Fluor 488 Goat anti-Chicken IgY H&L (1:500, ab150169, Abcam), Alexa Fluor 594 Donkey anti-Mouse IgG (H + L) (1:500, A-21203, Thermo Fisher Scientific), and Alexa Fluor 647 Donkey anti-Rabbit IgG (H + L) (1:500, A-31573, Thermo Fisher Scientific). The number of Bassoon or Homer1 puncta was counted alongside each neurite of 50 μm in length. The cell nuclei were stained with Hoechst 33342 (1:400, H-1399, Thermo Fisher Scientific). Cell images were acquired on an Olympus IX-81 FV1000 confocal microscope (Olympus) using a 63x water immersion objective lens and Olympus Fluoview software (Version 4.2a). Only representative images are shown.

Chen Z.S., Ou M., Taylor S., Dafinca R., Peng S.I., Talbot K, & Chan H.Y. (2023). Mutant GGGGCC RNA prevents YY1 from binding to Fuzzy promoter which stimulates Wnt/β-catenin pathway in C9ALS/FTD. Nature Communications, 14, 8420.

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Immunocytochemistry Protocol for HEK293 and SK-N-MC Cells

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The HEK293 or SK-N-MC cells were seeded on coverslips for 48 h (Marienfeld-Superior, Lauda-Königshofen, Germany). For the immunocytochemistry experiment shown in Fig. 6a, HEK293 cells were used and transfection lasted 24 h. For the rest of the immunocytochemistry experiments in this study, SK-N-MC cells were used and transfection lasted 48 h. Transfected cells were fixed with 3.7% paraformaldehyde for 15 min followed by permeabilization with 0.1% Triton X-100 for another 15 min. The cells were blocked with 5% goat serum at 25 °C for 1 h, followed by the incubation with primary antibody at 4 °C for 16 h. The cells were then washed three times with 1× PBS each for 5 min. The secondary antibody was used to incubate cells at 25 °C for 1 h. The cells were then washed five times with 1× PBS each for 5 min. The primary and secondary antibodies used were anti-HA (1:200; H3663, Sigma-Aldrich) and Alexa Fluor^® 488 AffiniPure goat anti-mouse IgG (H + L) (1:400; 115-545-062, Jackson ImmunoResearch, West Grove, PA, USA). The cell nuclei were stained with Hoechst 33342 (1:400; H-1399, Thermo Fisher Scientific) at 25 °C for 5 min. Cell images were acquired using a Zeiss LSM confocal microscope (Zeiss, Oberkochen, Germany), and images were analyzed using Fiji software (Version 2.0.0-rc-69/1.52n, NIH).

Chen Z.S., Huang X., Talbot K, & Chan H.Y. (2021). A fine balance between Prpf19 and Exoc7 in achieving degradation of aggregated protein and suppression of cell death in spinocerebellar ataxia type 3. Cell Death & Disease, 12(2), 136.

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Immunofluorescence Assay for Taxane-Resistant TNBC

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For immunofluorescence assay, parental and taxane-resistant TNBC cells were seeded in 6-well plates and treated with UNC1999 (3 μmol/L), UNC2400 (3 μmol/L), or DMSO on the following day for 4 days. Cells were then counted and seeded into 24-well plates at a density of 1 × 10 5 cells per well and treated again with UNC1999 (3 μmol/L), UNC2400 (3 μmol/L), or DMSO for an additional 24 hours. Cells were fixed in methanol and stained overnight with J2 scion antibody for dsRNA staining (Mouse J2 antibody Product no: 10010200 Scicons 1:500) or for nuclear staining (Hoescht, H1399 Thermo Fisher Scientific, 1:2,000), incubated in secondary antibody (Cell Signaling Technology 4410S anti-mouse IgG Alexa 647 1:1000) or anti-Rabbit secondary antibody (Cell Signaling Technology, Alexa 488 1:1,000), and imaged using a 40× objective. Total and dsRNA-positive cells in a minimum of 4 independent fields (at least 1,000 total cells per condition) were quantified. For IHC, tumors were harvested from mice, and 5-μm sections of formalin-fixed, paraffin-embedded murine mammary tumors were IHC stained with H3K27me3 antibody (Diagenode; 1:100 dilution).

Deblois G., Tonekaboni S.A.M., Grillo G., Martinez C., Kao Y.I., Tai F., Ettayebi I., Fortier A.M., Savage P., Fedor A.N., Liu X., Guilhamon P., Lima-Fernandes E., Murison A., Kuasne H., Ba-Alawi W., Cescon D.W., Arrowsmith C.H., De Carvalho D.D., Haibe-Kains B., Locasale J.W., Park M, & Lupien M. (2020). Epigenetic Switch-Induced Viral Mimicry Evasion in Chemotherapy-Resistant Breast Cancer. Cancer discovery, 10(9).

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Immunofluorescence Staining of Mitochondrial Proteins

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Cells were seeded on permanox chamber slides (Thermo Scientific, 177429) and fixed with 4% formaldehyde (Ted Pella, Inc., 18505) in phosphate-buffered saline [PBS] (Gibco, 14200–075) for 10 min and then washed in PBS. Cells were then permeabilized in PBS with 0.25% Triton X-100 (Sigma-Aldrich, T9284) for 10 min. Cells were washed in PBS and blocked in PBS with 5% goat serum (Sigma-Aldrich, G9023). Cells were incubated in primary antibody diluted in PBS with 5% goat serum overnight at 4°C. Primary antibodies used were as follows: MAP1LC3A (1:100, Cell Signaling Technology, 4108), OPA1 (1:100, BD Transduction Labs, 612607), TOMM70A (1:200, Proteintech, 14528–1-AP), PPARGC1A (1:100, Santa Cruz Biotechnology, sc-13067). Cells were washed in PBS and incubated in corresponding fluorescent secondary antibodies (1:100, Life Technologies, A11032, A11034, A21207) and Hoechst 33342 nuclear stain (1:1000; Life Technologies, H1399) for 1 h in the dark at room temperature. Cells were washed and coverslipped with hardset Vectashield mounting medium (Vector Labs, H-1400). Imaging was performed using a Keyence BZ-9000 microscope (Keyence; Osaka, Japan).

Sin J., Andres A.M., Taylor D.J., Weston T., Hiraumi Y., Stotland A., Kim B.J., Huang C., Doran K.S, & Gottlieb R.A. (2015). Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts. Autophagy, 12(2), 369-380.

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