Dapi 4 6 diamidino 2 phenylindole

Manufactured by Merck Group

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DAPI (4′,6-diamidino-2-phenylindole) is a fluorescent dye used in molecular biology and microscopy applications. It binds strongly to DNA and emits blue fluorescence when excited by ultraviolet light. DAPI is commonly used to stain and visualize cell nuclei.

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Lab products found in correlation

Triton x 100, by Merck Group (21 mentions) Bovine serum albumin (bsa), by Merck Group (14 mentions) Alexa fluor 488, by Thermo Fisher Scientific (13 mentions) Paraformaldehyde (pfa), by Merck Group (11 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions) Lsm 700, by Zeiss (6 mentions) Alexa fluor 594, by Thermo Fisher Scientific (6 mentions) Lsm 710, by Zeiss (6 mentions) Tritc conjugated phalloidin, by Merck Group (5 mentions) Vectashield, by Vector Laboratories (5 mentions) Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (5 mentions) Tcs sp5 2, by Leica (4 mentions) Prolong gold antifade mountant, by Thermo Fisher Scientific (4 mentions) Confocal laser scanning microscope, by Leica (4 mentions) Lsm 780, by Zeiss (4 mentions) Paraformaldehyde solution, by Merck Group (3 mentions) Fluorescent microscope, by Leica (3 mentions) Triton x 100, by Thermo Fisher Scientific (3 mentions) Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (3 mentions) Propidium iodide, by Merck Group (3 mentions)

348 protocols using dapi 4 6 diamidino 2 phenylindole

Cell Cytoskeleton Staining Protocol

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The
detailed method has
already been reported by our group. Adhered cells on samples were
fixed with a 4% paraformaldehyde solution (Sigma-Aldrich, Milwaukee,
WI) for 20 min, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich,
WI, Milwaukee) for 15 min, and stained with TRITC-conjugated phalloidin
(Millipore, Billerica, MA) for 1 h and 4,6-diamidino-2-phenylindole
(DAPI; Millipore, Billerica, MA) for 5 min. After each process, the
samples were washed three times with 1× PBS for 5 min each. The
images of the stained cells were taken using a fluorescence microscope
(Zeiss, Germany).

Kim W., Kim G.E., Attia Abdou M., Kim S., Kim D., Park S., Kim Y.K., Gwon Y., Jeong S.E., Kim M.S, & Kim J. (2020). Tendon-Inspired Nanotopographic Scaffold for Tissue Regeneration in Rotator Cuff Injuries. ACS Omega, 5(23), 13913-13925.

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Infarct Size and BMC Retention Analysis

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For fixed tissues, paraffin blocks were cut into 5-μm-thick sections and were mounted on glass slides for staining. Slides were deparaffinized and underwent antigen retrieval in hot citric acid buffer. Stains were conducted against the following proteins: α-sarcomeric actin (A2547, Sigma; St. Louis, MO) and EGFP (ab111258, Abcam; Cambridge, MA). The following secondary antibodies were purchased from Jackson Immunoresearch Laboratories (West Grove, PA) and used for detection of primary antibodies as follows: rhodamine red-X donkey anti-Mouse IgM (715-295-020) was used to detect α-sarcomeric actin; and FITC donkey anti-goat IgG (705-095-147) was used to detect EGFP. Nuclei in embedded tissues were stained with 4′,6-diamidino-2-phenylindole (DAPI, Millipore; Billerica, MA). Confocal micrographs of all immunostains were acquired using a Nikon Eclipse T1 confocal microscope (Nikon Inc.; Melville, NY). Infarct size was determined with Masson’s trichrome staining. GFP⁺ BMC retention was quantified with fluorescent microscopy assessed from fixed area microscope fields from basal sections of heart and expressed as number of GFP⁺ BMC’s per field. For infarcted animals, Ki67⁺ nuclei and GFP⁺ BMC’s were counted in the infarct zone, the infarct border zone, and in the distal myocardium (1000 μm from the infarct edge).

Chirico E.N., Ding D., Muthukumaran G., Houser S.R., Starosta T., Mu A., Margulies K.B, & Libonati J.R. (2015). Acute aerobic exercise increases exogenously infused bone marrow cell retention in the heart. Physiological Reports, 3(10), e12566.

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Cytoskeleton and Focal Adhesion Visualization

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Immunofluorescent staining
was performed to observe the cytoskeleton or FAs. After incubation,
cells seeded on the gels were fixed with 4% paraformaldehyde (Sigma)
for 10 min and treated in 0.2% Triton X-100 (Sigma) for 10 min at
room temperature. For cytoskeleton staining, samples were incubated
with phalloidin-Atto 633 (Sigma) and 4',6-diamidino-2-phenylindole
(DAPI) (Millipore) for 1 h; for FAs staining, nonspecific binding
sites were blocked in 10% BSA solution for 1 h first, followed by
incubation with primary antibody antivinculin (Abcam, ab18058) and
anti-integrin β1 antibody (ab30394) for 1 h and, subsequently,
with secondary antibody Alexa 488 goat anti-mouse (Thermo Fisher Scientific,
R37120), phalloidin, and DAPI for 1 h. The stained cells were imaged
using the SP8, and spreading areas and perimeter measurements were
obtained using Fiji’s in-built “Measure” function
after drawing a region of interest around cells. More than 80 cells
were measured in three separate experiments.

Xie J., Bao M., Bruekers S.M, & Huck W.T. (2017). Collagen Gels with Different Fibrillar Microarchitectures Elicit Different Cellular Responses. ACS Applied Materials & Interfaces, 9(23), 19630-19637.

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Immunostaining and TUNEL Assay for Cardiac Apoptosis

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Slides underwent deparaffinization for antigen retrieval with citrate buffer. The following primary antibodies were used: α-sarcomeric actin (A2172, Sigma-Aldrich; St. Louis, MO), sarcomeric tropomyosin (Sigma-Aldrich #T9283; St Louis, MO). Secondary antibodies were used to detect the primary antibodies as follows: rhodamine red-X donkey anti-mouse IgM (Jackson Immunoresearch Laboratories; West Grove, PA) to detect α-sarcomeric actin or sarcomeric tropomyosin. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI, Millipore; Billerica, MA). EdU detection was performed using the EdU Click-iT Imaging kit (Life Technologies; Carlsbad, CA).
Immunostaining with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) was performed on paraffin-embedded heart tissues obtained from12 weeks old wild-type male C57BL/6 mice. These mice were subcutaneously injected with 200μl of physiological solution saline and sacrificed 24 hours later. No heart injury is expected to be obtained after saline injections. The DeadEnd Fluorometric TUNEL system was used to label apoptotic nuclei (Promega #G3250; Madison, WI). For positive control slides were treated with DNASE I (10U/l) prior to TUNEL staining.

Gross P., Honnorat N., Varol E., Wallner M., Trappanese D.M., Sharp T.E., Starosta T., Duran J.M., Koller S., Davatzikos C, & Houser S.R. (2016). Nuquantus: Machine learning software for the characterization and quantification of cell nuclei in complex immunofluorescent tissue images. Scientific Reports, 6, 23431.

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Quantifying Immunosuppressive MDSC Subsets in Breast Cancer

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Immunofluorescence was performed on 30 cases of formaldehyde-fixed, paraffin-embedded breast cancer tissues. Tissue sections were incubated with rabbit anti-human IDO (Millipore Corporation, Billerica, MA), rabbit anti-phospho-STAT3 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-human NIK (Santa Cruz biotechnology, CA, USA) and mouse anti-CD33 (Abcam, San Francisco, CA) antibodies overnight at 4°C, and Alexa Fluor^® goat anti-mouse or anti-rabbit IgG secondary antibodies for 1 hour. 4′-6-diamidino-2-phenylindole (DAPI, Millipore Corporation, Billerica, MA) was used for nuclear counterstaining. Images were acquired using a Leica SP2 confocal microscope (Leica, Mannheim, Germany).
Breast cancer tissue sections were immunostained with phospho-STAT3 (pSTAT3) and CD33 for pSTAT3⁺ MDSCs, IDO and CD33 for IDO⁺ MDSCs, as well as NIK and CD33 for NIK⁺ MDSCs. Five representative high-power fields (×400 magnification) for each tissue section were selected for histology evaluation, and the percentages of pSTAT3⁺MDSCs, IDO⁺MDSCs and NIK⁺MDSCs in MDSCs were count and analyzed.

Yu J., Wang Y., Yan F., Zhang P., Li H., Zhao H., Yan C., Yan F, & Ren X. (2014). Noncanonical NF-κB Activation Mediates STAT3-stimulated IDO Upregulation in Myeloid-Derived Suppressor Cells in Breast Cancer. Journal of immunology (Baltimore, Md. : 1950), 193(5), 2574-2586.

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Visualizing Actin Cytoskeleton in Cardiomyocytes

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Rhodamine-phalloidin was used to visualize actin fibers under fluorescence microscopy. HCMs were stained with rhodamine-phalloidin (1 : 100 dilute, Millipore) to target F-actin in the cytosol and then stained with 4′,6-diamidino-2-phenylindole (DAPI) (1 : 10,000 dilution, Millipore) for 5 min to target DNA in the cell nucleus. HCMs were viewed under a camera attached to a microscope. The surface area was determined using image analysis software (MetaMorph Imaging System, Meta Imaging Series 4.5) and calculated as the mean of 100 cells from at least ten randomly chosen fields (×40 objective) in three separate experiments.

Jen H.L., Liu P.L., Chen Y.H., Yin W.H., Chen J.W, & Lin S.J. (2016). Peroxisome Proliferator-Activated Receptor α Reduces Endothelin-1-Caused Cardiomyocyte Hypertrophy by Inhibiting Nuclear Factor-κB and Adiponectin. Mediators of Inflammation, 2016, 5609121.

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Visualizing VEGFR2 Expression in HUVECs

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HUVEC cells were seeded on a slide in the 24-well plate. Immunofluorescence images of VEGFR2 expression on HUVECs were captured, after treatment with indicated media (Peptostreptococcus anaerobius treated macrophage media) stimulation for 72h. Cells were fixed with 10% paraformaldehyde, the VEGFR were stained by FITC-VEGFR antibody, and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Millipore) for 10 min at 4°C. Images were taken using a Leica fluorescent microscope and a TCS SP5 confocal laser scanning microscope (Leica Microsystems, Wetzlar, GER).

Zhou G., Zhou F., Gu Y., Zhang M., Zhang G., Shen F., Hua K, & Ding J. (2022). Vaginal Microbial Environment Skews Macrophage Polarization and Contributes to Cervical Cancer Development. Journal of Immunology Research, 2022, 3525735.

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Quantifying Neurite Outgrowth in DRG Neurons

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DRG neuron cultures were prepared from 8-week-old male SD rats as previously described [15 (link)]. DRG neurons were cultured for 24 h in serum-free medium (DMEM/F12 supplemented with B27 (Invitrogen)) for use in neurite outgrowth with DPSC-CM. DRG neurons were immunostained with rabbit polyclonal anti-neurofilament heavy-chain antibody (Millipore). Alexa Fluor 488-coupled goat anti-rabbit IgG antibody (Invitrogen) was applied as the second antibody. Coverslips were counterstained with 4’ ,6-diamidino-2-phenylindole (DAPI; Millipore). Neurite outgrowth was observed in 40–50 neurons per cover slip, and the total length and joint number of neurites was calculated by a computed image analysis system (Angiogenesis Image Analyzer Ver. 2, KURABO Industries, Osaka, Japan).

Omi M., Hata M., Nakamura N., Miyabe M., Ozawa S., Nukada H., Tsukamoto M., Sango K., Himeno T., Kamiya H., Nakamura J., Takebe J., Matsubara T, & Naruse K. (2017). Transplantation of dental pulp stem cells improves long-term diabetic polyneuropathy together with improvement of nerve morphometrical evaluation. Stem Cell Research & Therapy, 8, 279.

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Cytoskeletal Organization on Hybrid Nanofiber Scaffolds

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To evaluate cellular cytoskeletal organization on the pristine PLGA, nHA/PLGA, and BMP-g-nHA/PLGA hybrid nanofiber scaffolds, double staining was performed according to the previously reported method [22 (link)]. Briefly, osteoblast cells were seeded onto the nanofiber scaffolds (2 × 10⁴ cells/mL) separately and cultured for 3 days. The cells were then fixed with 4% paraformaldehyde in PBS. After fixation, the samples were washed using a PBS containing 0.05% Tween-20. The samples were permeabilized with 0.1% Triton X-100 in PBS for 15 min at 25°C and then incubated for 30 min in a PBS containing 1% bovine serum albumin (BSA). This was followed by the addition of 5(6)-tetramethyl-rhodamine isothiocyanate-conjugated phalloidin (TRITC Millipore) for approximately 1 h. The nanofiber scaffolds were washed three times (10 min each) using the buffer solution and incubated with 4′,6-diamidino-2-phenylindole (DAPI, Millipore) for 5 min. Fluorescence images were visualized using a confocal laser-scanning microscope (Model 700; Carl Zeiss, Oberkochen, Germany).

Haider A., Kim S., Huh M.W, & Kang I.K. (2015). BMP-2 Grafted nHA/PLGA Hybrid Nanofiber Scaffold Stimulates Osteoblastic Cells Growth. BioMed Research International, 2015, 281909.

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Cytoskeletal Analysis of Osteoblast Cells on PLGA/nHA Scaffolds

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To evaluate the cytoskeletal organization of cells onto the PLGA/nHA-I and PLGA/nHA composite as well as pristine PLGA nanofiber scaffolds, double staining was performed according to the manufacturer's protocol. Briefly, osteoblast cells were seeded onto the scaffolds (2 × 10⁴ cells/mL) and were cultured for 3 days. The cells were fixed with 4% paraformaldehyde in PBS. After fixation, the samples were washed using PBS buffer solution containing (0.05% Tween-20). The samples were permeabilized with 0.1% Triton X-100 in PBS for 15 min at 25°C and then incubated for 30 min in PBS containing 1% bovine serum albumin (BSA). This was followed by the addition of 5(6)-tetramethyl-rhodamine isothiocyanate-conjugated phalloidin (Millipore) (TRITC) for approximately 1 h. The samples were washed three times (10 min each) using the buffer solution and incubated with 4′,6-diamidino-2-phenylindole (DAPI) (Millipore) for 5 min. Fluorescence images were visualized using a confocal laser scanning microscope (model 700; Carl Zeiss, Oberkochen, Germany) after washing the scaffolds three times (10 min each) with the buffer solution.

Haider A., Gupta K.C, & Kang I.K. (2014). PLGA/nHA hybrid nanofiber scaffold as a nanocargo carrier of insulin for accelerating bone tissue regeneration. Nanoscale Research Letters, 9(1), 314.

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