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Dapi stain

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DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain that binds strongly to adenine-thymine (A-T) rich regions in DNA. It is commonly used in fluorescence microscopy to visualize and locate the nucleus of cells.

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Lab products found in correlation

Lsm 700 confocal microscope, by Zeiss (2 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) P tbk1 ser172, by Cell Signaling Technology (2 mentions) Immunofluorescence kit, by Cell Signaling Technology (2 mentions) Lysotracker red dnd 99, by Thermo Fisher Scientific (2 mentions) Calnexin, by Abcam (2 mentions) Facsaria 2, by BD (2 mentions) Lsm 510, by Zeiss (2 mentions) Mouse monoclonal anti cre antibody, by Abcam (2 mentions) Axio observer microscope, by Zeiss (1 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions) Micro cover glass, by Avantor (1 mentions) Microscope slide, by Thermo Fisher Scientific (1 mentions) Alexa488 anti rabbit antibody, by Thermo Fisher Scientific (1 mentions) Vectashield, by Vector Laboratories (1 mentions) 24 well plate, by Greiner (1 mentions) Collagenase, by Thermo Fisher Scientific (1 mentions) Alexafluor 546 anti mouse igg, by Thermo Fisher Scientific (1 mentions) Hepatocyte growth factor (hgf), by Merck Group (1 mentions)

33 protocols using dapi stain

1

Quantifying Intracellular Parasites in HFF Cells

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Wells with coverslips were fixed with 4% paraformaldehyde for ten minutes. The coverslips were washed five times with PBS, mounted onto microscope slides with ProLong Gold antifade reagent along with DAPI stain (Life Technologies) and allowed to set overnight at room temperature in the dark. Parasites were counted at 100x oil immersion on a Zeiss AxioObserver Microscope. One hundred cells on each coverslip were counted, with three coverslips per timepoint. Percent infected HFF cells and parasite burden per infected cell were computed.
Houston-Ludlam G.A., Belew A.T, & El-Sayed N.M. (2016). Comparative Transcriptome Profiling of Human Foreskin Fibroblasts Infected with the Sylvio and Y Strains of Trypanosoma cruzi. PLoS ONE, 11(8), e0159197.
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2

Quantifying MLKL Phosphorylation in HT-29 Cells

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HT-29 cells were seeded onto micro cover glass (VWR) in 24-well plates (Greiner Bio-One) at 180,000 cells per well. Twelve hours after seeding, TSZ was added to appropriate wells, and samples were fixed 6 hours post-treatment with 4% paraformaldehyde (Sigma) for 30 min at RT. Anti-MLKL phospho S358 antibody (1:200) was added to each sample in immunofluorescence blocking buffer (PBS with 3% BSA, 1% saponin, and 1% Triton X-100) and incubated at 4°C overnight. After washing 5× with 1× phosphate buffered saline (PBS), Alexa488 anti-rabbit antibody (1:500) (Life Technologies), DAPI stain (1:300), and Alexa660 phalloidin (1:50) (Life Technologies) in immunofluorescence blocking buffer were added to each sample and incubated for 1 hour at RT. Samples were washed 5× with 1× PBS. micro cover glasses were then mounted onto microscope slides (Fisher Scientific) with Vectashield (Vector Laboratories Inc). Images were taken with Zeiss LSM 700 confocal microscope and processed with Velocity software.
Dovey C.M., Diep J., Clarke B.P., Hale A.T., McNamara D.E., Guo H., Brown NW J.r., Cao J.Y., Grace C.R., Gough P.J., Bertin J., Dixon S.J., Fiedler D., Mocarski E.S., Kaiser W.J., Moldoveanu T., York J.D, & Carette J.E. (2018). MLKL requires the inositol phosphate code to execute necroptosis. Molecular cell, 70(5), 936-948.e7.
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3

Hepatocyte Cell Culture Protocol

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Glass slides (75 × 25 mm2) were obtained from VWR (West Chester, PA). Thiol-silane was purchased from Gelest, Inc. (Morrisville, PA). Poly(dimethylsiloxane) (PDMS) was acquired from Dow Corning (Midland, MI). Collagenase, collagen from rat tail (type I), AlexaFluor 488 anti-sheep IgG and AlexaFluor 546 anti-mouse IgG were purchased from Invitrogen (Carlsbad, CA). Hepatocyte growth factor (HGF) and transforming growth factor-β1 (TGF-β1) were obtained from Sigma– Aldrich (St. Louis, MO). Sheep anti-rat albumin antibody was purchased from Bethyl Laboratories (Montgomery, TX). Phosphate-buffered saline (1× PBS), Duelbecco's modified eagle medium (DMEM), sodium pyruvate, DAPI stain and fetal bovine serum (FBS) were purchased from Life technologies. Paraformaldehyde was purchased from Electron Microscopy Sciences (Hatfield, PA). SU11274 was purchased from Selleckchem.
Patel D., Haque A., Gao Y, & Revzin A. (2015). Using Reconfigurable Microfluidics to Study the Role of Hepatocyte Growth Factor in Autocrine and Paracrine Signaling of Hepatocytes. Integrative biology : quantitative biosciences from nano to macro, 7(7), 815-824.
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4

Microscopic Analysis of BCL7B Knockdown

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KATOIII cells were grown on 2-well chamber slides (Lab-Tek, Campbel, CA) at 1×105 cells/well for approximately 24 h prior to transfection. Forty-eight hours after transfection with control-siRNA or BCL7B-siRNA, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.1% Triton X-100 for 3 min. Then, the cells were washed and incubated in 0.1 µg/mL DAPI stain (Life Technology, Carlsbad, CA) overnight at room temperature in the dark. Samples were observed under a fluorescence microscope (with a UV filter), and acquired images were digitally analyzed with ImageJ software (National Institutes of Health, Bethesda, MA).
Uehara T., Kage-Nakadai E., Yoshina S., Imae R, & Mitani S. (2015). The Tumor Suppressor BCL7B Functions in the Wnt Signaling Pathway. PLoS Genetics, 11(1), e1004921.
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5

Dissociation and Isolation of Tumor Cells

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Tumors were dissected into small pieces and re-suspended in digestion buffer (RPMI (Gibco) containing 100 U ml−1 Collagenase IV (Sigma), 500 U ml−1 Collagenase II (Sigma) and 0.2 mg ml−1 DNase I (Sigma)). Samples were incubated on a shaker at 37 °C for 40 min and vigorously shaken at the 20 and 40 min mark. Samples were passed through a 70 µM cell strainer (Fisher Scientific) and pelleted at 800×g for 2 min. Samples were re-suspended in FACS buffer (PBS containing 2% FBS (Gibco)) with 1 µg ml−1 DAPI stain (Life Technologies). Approximately 100,000 live cells were sorted into PBS + 0.04% BSA.
Mahmood M., Liu E.M., Shergold A.L., Tolla E., Tait-Mulder J., Huerta-Uribe A., Shokry E., Young A.L., Lilla S., Kim M., Park T., Boscenco S., Manchon J.L., Rodríguez-Antona C., Walters R.C., Springett R.J., Blaza J.N., Mitchell L., Blyth K., Zanivan S., Sumpton D., Roberts E.W., Reznik E, & Gammage P.A. (2024). Mitochondrial DNA mutations drive aerobic glycolysis to enhance checkpoint blockade response in melanoma. Nature Cancer, 5(4), 659-672.
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6

Nile Red Staining of Adipocytes

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Nile red stain was prepared following the protocol of Greenspan et al. [60 (link)] with minor modifications. Nile red was prepared by dissolving 5 mg Nile red powder (Sigma-Aldrich N3013, USA) in 5 mL of acetone to obtain 1 mg/mL stock solution. Nile red stock solution was diluted in 1mM trizma-maleate (Sigma-Aldrich T3128, USA) and 3% w/v Polyvinylpyrrolidone (Sigma-Aldrich P2307, USA) to obtain 1:100 Nile red stain solution. Differentiation media was discarded, the adipocytes cells were washed with PBS and fixed with 4% paraformaldehyde for 1 h. Fixed cells were stained directly with 1:100 Nile red stain solution and DAPI stain (Life Technologies P36930, Eugene, OR, USA). Stained cells were imaged under the Olympus fluorescent microscope using Cellsens standard software.
Fayyad A.M., Khan A.A., Abdallah S.H., Alomran S.S., Bajou K, & Khattak M.N. (2019). Rosiglitazone Enhances Browning Adipocytes in Association with MAPK and PI3-K Pathways During the Differentiation of Telomerase-Transformed Mesenchymal Stromal Cells into Adipocytes. International Journal of Molecular Sciences, 20(7), 1618.
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7

Immunofluorescence Staining of Adherent Cells

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Cells on coverslips were fixed in 4% PFA in PBS supplemented with 4% sucrose for 15 min at room temperature. After extensive washing with PBS, cells were blocked and permeabilized with 10% normal goat serum/0.1% Triton X-100 in PBS. For surface labeling, blocking solution without detergent was used. Cells were incubated with primary antibodies for 1–3 h at room temperature or overnight at +4°C with gentle rocking and for 1 h with donkey or goat secondary antibodies conjugated to Alexa or DyLight fluorophores (Thermo Fisher Scientific or Jackson Immunoresearch). DAPI stain was included to label nuclei (Thermo Fisher Scientific). Coverslips were again washed with PBS, dipped in water, and mounted on glass slides using Mowiol/DABCO mounting medium.
Ahmad T., Vullhorst D., Chaudhuri R., Guardia C.M., Chaudhary N., Karavanova I., Bonifacino J.S, & Buonanno A. (2022). Transcytosis and trans-synaptic retention by postsynaptic ErbB4 underlie axonal accumulation of NRG3. The Journal of Cell Biology, 221(7), e202110167.
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8

Brightfield, Epifluorescence, and Confocal Imaging of Cells

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Brightfield and epifluorescence images were taken using a Nikon Eclipse Ti Microscope, Hamamatsu Photonics K.K. C10600-10B-H camera, 10× objective lens, and native NIS-Elements AR 5.02.00 software. Samples were submerged in PBS during image acquisition. Nuclei were labeled with a 4′,6-diamidino-2-phenylindole (DAPI) stain (Sigma-Aldrich) and GFAP or Tuj1 were labeled with either a 488 or a 633 dye-conjugated secondary antibody (Thermo-Fisher). Confocal images were taken using a Prairie Technologies 2-photon and confocal microscope, QuantEM 512SC camera, 60X objective lens, and native Prairie View software. Samples were submerged in PBS during image acquisition. Nuclei were labeled with a DAPI stain, F-actin was labeled with an Alexa Fluor 546 Phalloidin (Thermo-Fisher), and other targets were labeled with either a 488 or 633 dye–conjugated secondary antibody. All image processing and analysis was carried out in the free ImageJ software.
Kang P.H., Schaffer D.V, & Kumar S. (2020). Angiomotin links ROCK and YAP signaling in mechanosensitive differentiation of neural stem cells. Molecular Biology of the Cell, 31(5), 386-396.
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9

Decellularized Skeletal Muscle Scaffold Cellularity

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Immediately after the decellularization of the skeletal muscle, DSMG samples were stained with DAPI stain (Thermo Fisher Scientific,USA), following the manufacturer’s sample preparation guidelines. They were then examined under a fluorescent microscope (Nikon®, Eclipse Ti2 inverted microscope) with an excitation filter of 355–425 nm to assess the cellularity of the scaffolds.
hakeem L., Al-Kindi M., AlMuraikhi N., BinHamdan S, & Al-Zahrani A. (2021). Evaluation of the regenerative potential of decellularized skeletal muscle seeded with mesenchymal stromal cells in critical-sized bone defect of rat models. The Saudi Dental Journal, 33(5), 248-255.
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10

Praziquantel Liposomal Formulation Development

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Praziquantel, phospholipids (Asolectin from soybean), cholesterol (CHOL), phosphatidylethanolamine distearoyl methoxypolyethleneglycol conjugate (DSPE-mPEG2000COOH), N-hydroxysulfosuccinimide (NHS), N,N′ -dicyclohexylcarbodiimide (DCC), and 49,409 Atto 488 Phalloidin and Biochemical assays (AST, ALT, ALP, bilirubin, and creatinine) kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). DAPI stain was purchased from Thermofisher. Monoclonal calpain antibody was purchased from Abcam (ab154167; Cambridge, UK) raised from rabbit; RAW 264.7 murine macrophage and 3T3 human fibroblast cell lines were procured from ATCC (Manassas, VA, USA). All other chemicals used in the study were of analytical grade.
Adekiya T.A., Kumar P., Kondiah P.P, & Choonara Y.E. (2022). In Vivo Evaluation of an Antibody-Functionalized Lipoidal Nanosystem for Schistosomiasis Intervention. Pharmaceutics, 14(8), 1531.
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