The A549 cell monolayers were fixed (methanol, 10 min), rehydrated with PBS and permeabilized using 0.1% Triton X-100 solution for 20 min. Monolayers were incubated with primary rabbit anti-N protein (1:500; Sigma, Darmstadt, Germany) and mouse anti-Gc protein (1:50; Abcam, Waltham, MA, USA) antibodies (3 h, RT) followed by donkey anti-rabbit IgG (H + L) Alexa Fluor 647 (1:1000, Invitrogen, Rockford, USA) and donkey anti-mouse IgG (H + L) Alexa Fluor 555 (1:1000, Invitrogen, Rockford, USA), respectively. The nucleus was visualized using 4′,6-diamidino-2-phenylindol (DAPI; Sigma, Darmstadt, Germany). Images were captured using laser confocal microscope LSM 700 (Carl Zeiss) and ZEN software.
4 6 diamidino 2 phenylindol dapi
Manufactured by Merck Group
Sourced in United States, Germany
4',6-diamidino-2-phenylindol (DAPI) is a fluorescent dye used to stain and visualize nucleic acids, particularly DNA, in biological samples. It preferentially binds to adenine-thymine (A-T) rich regions in the minor groove of DNA. When excited by ultraviolet (UV) or blue light, DAPI emits a blue fluorescence that can be detected using fluorescence microscopy or flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (5 mentions)
Blasticidin, by Thermo Fisher Scientific (4 mentions)
Superscript 2 rt kit, by Thermo Fisher Scientific (4 mentions)
Axio observer z1, by Zeiss (3 mentions)
Lsm 900 confocal microscope, by Zeiss (3 mentions)
Triton x 100, by Merck Group (2 mentions)
Pfu 2 high fidelity polymerase, by Agilent Technologies (2 mentions)
Ts100f fluorescent microscope, by Nikon (2 mentions)
Pfu 2 high delity polymerase, by Agilent Technologies (2 mentions)
Ts100f uorescent microscope, by Nikon (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Alexa fluor 594 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Donkey anti mouse igg h l alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Mouse anti gc protein, by Abcam (1 mentions)
Bx53 fluorescent microscope, by Olympus (1 mentions)
Ferric ammonium citrate, by Merck Group (1 mentions)
Deaprotinin, by Merck Group (1 mentions)
Enhanced chemiluminescence detection kit, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
24 protocols using 4 6 diamidino 2 phenylindol dapi
1
Immunofluorescence Imaging of SARS-CoV-2 Proteins
2
Quantifying Actin Stress Fiber Orientation
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Cells were first fixed
with 4% paraformaldehyde (PFA) for 20 min
and then a permeabilization buffer containing phosphate-buffered saline
(PBS), 2 mg/mL Bovine Serum Albumin (BSA), and 0.1% Triton X-100.
Then, cells were incubated with phalloidin (Biotium, CA, USA) for
1 h at room temperature. Finally, cells were washed three times with
the permeabilization buffer and incubated for 2 min with 4′,6-
Diamidino-2-Phenylindol (DAPI; Sigma-Aldrich, St. Louis, MO, USA).
All coverslips were then mounted on a slide and observed under an
Olympus BX53 fluorescent microscope equipped with an XM10 Monochrome
CCD camera (Olympus Corp., Japan). For the characterization of the
actin stress fibers, the FilamentSensor tool (University of Göttingen,
Germany) was used.42 (link) In the reconstructed
images, each color corresponds to a different fiber orientation. Stress
fiber orientation was assessed using the order parameter S = cos2θ, where the higher the value of S,
the more oriented the fibers become. Cell elongation was assessed
by using optical microscopy images. ImageJ software was used to automatically
measure factor E from cells, which equals the long
axis divided by the short axis minus one. Thus, E is zero for a circle and one for an ellipse with an axis ratio of
1:2. The cells that presented E values 0–0.5
were considered as spherical, 0.5–1 as ellipsoid, and E values higher than 1 as elongated.
with 4% paraformaldehyde (PFA) for 20 min
and then a permeabilization buffer containing phosphate-buffered saline
(PBS), 2 mg/mL Bovine Serum Albumin (BSA), and 0.1% Triton X-100.
Then, cells were incubated with phalloidin (Biotium, CA, USA) for
1 h at room temperature. Finally, cells were washed three times with
the permeabilization buffer and incubated for 2 min with 4′,6-
Diamidino-2-Phenylindol (DAPI; Sigma-Aldrich, St. Louis, MO, USA).
All coverslips were then mounted on a slide and observed under an
Olympus BX53 fluorescent microscope equipped with an XM10 Monochrome
CCD camera (Olympus Corp., Japan). For the characterization of the
actin stress fibers, the FilamentSensor tool (University of Göttingen,
Germany) was used.42 (link) In the reconstructed
images, each color corresponds to a different fiber orientation. Stress
fiber orientation was assessed using the order parameter S = cos2θ, where the higher the value of S,
the more oriented the fibers become. Cell elongation was assessed
by using optical microscopy images. ImageJ software was used to automatically
measure factor E from cells, which equals the long
axis divided by the short axis minus one. Thus, E is zero for a circle and one for an ellipse with an axis ratio of
1:2. The cells that presented E values 0–0.5
were considered as spherical, 0.5–1 as ellipsoid, and E values higher than 1 as elongated.
3
Immunofluorescence Staining of Cell Markers
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Cells were washed twice with PBS and fixed in 4% paraformaldehyde (PFA: Wako) at room temperature for 10 min. After washing with PBS, cells were permeabilized with PBS containing 0.1% Triton X-100 for 10 min, blocked with PBS containing 2% bovine serum albumin for 20 min, and incubated with an antibody against CD31 (clone: JC70A, Dako) and von Willebrand factor (vWF: Cat No. A0082, Dako) at 4°C overnight. Then, cells were incubated with AlexaFluor488- or Alexafluor594-conjugated secondary antibodies (1:1000; Life Technology) at room temperature for 1 hr. After washing, nuclei were labeled with 4’, 6-diamidino-2-phenylindol (DAPI: Sigma).
4
Cellular Apoptosis Pathway Analysis
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N-acetyl-cysteine (NAC), Ferric ammonium citrate (FAC), 2′, 7′-dichlorodihy-drofluorescein diacetate (H2DCF-DA), calcein-AM, Hoechst33258, 4′, 6-diamidino-2-phenylindol (DAPI), penicillin, streptomycin, leupeptin, pepstatin A, deaprotinin, phenylmethylsulfonylfluoride (PMSF), and 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES) were purchased from Sigma (St. Louis, MO, USA). Primary antibodies against cytochrome c, bcl-2, bax, cleaved Caspase-3, Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and beta-actin (β-actin) were purchased from Abcam (Cambridge, UK). AnnexinV–FITC/PI kit was obtained from KeyGen Biotech (Nanjing, China). Fetal bovine serum and alpha-modified Eagle’s medium (a-MEM) were purchased from Gibco (Waltham, MA, USA). Cell Mitochondria Isolation Kit, Enhanced Chemiluminescence detection kit, Western and immunoprecipitation (IP) Cell Lysis Kit, Bicinchoninic Acid Protein (BCA) Protein Assay Kit, and 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolcarbocyanineiodide (JC-1) were purchased from Beyotime (China). Cell Counting Kit-8 (CCK-8) assay kit was purchased from DojinDo (Japan).
5
Immunostaining of Endothelial Junctions
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Isolated vessels were fixed in ice-cold methanol for 10 min at room temperature. After washing with PBS, the vessels were blocked with 10% normal goat serum (Wako) in PBS supplemented with 0.1% Triton-X at room temperature for 1 h. After blocking, the cells were stained with a mouse anti-claudin-5 antibody (diluted 1/50; ThermoFisher Scientific) and a rabbit anti-platelet endothelial cell adhesion molecular-1 (PECAM-1) antibody (diluted 1/50; ThermoFisher Scientific) at 4 oC overnight. Then, the cells were washed with PBS and stained with an Alexafluor488-conjugated secondary antibody and an Alexafluor594-conjugated secondary antibody (diluted 1/500; ThermoFisher Scientific). After washing, the nuclei were labeled with 4’, 6-diamidino-2-phenylindol (DAPI; Sigma) for 10 min at room temperature. Images were obtained with a BZ-X700 microscope (KEYENCE).
6
Quantifying TGFB Signaling in Mammary Macrophages
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Macrophages positive for phosphorylated SMAD2, indicative of TGFB signalling [31 (link)], were detected by immunofluorescent staining of the formalin-fixed paraffin-embedded mammary glands. Following antigen retrieval, F4/80 antibody conjugated to Alexa-750 (Caltag Laboratories, Burlingame, CA, USA) and rabbit anti-pSMAD2 monoclonal antibody (Invitrogen) both at a 1:100 dilution were incubated with the slides overnight at 4 °C. Goat anti-rabbit antibody conjugated to Alexa-488 was used to detect pSMAD2 (1:500 dilution; 1 h at 4 °C) followed by 4′, 6-diamidino-2-phenylindol (DAPI) (Sigma, St Louis, USA) staining for 10 min at 1:1000 dilution. Images were captured and collected using an Olympus FV3000 Confocal Microscope at × 40 magnification. Macrophages were selected randomly for imaging, and the number of macrophages positive for pSMAD2 quantified and expressed as percent positive per mammary gland.
7
Migratory Ability of MenSC-eGFP Cells
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The migratory ability of MenSC-eGFP cells was determined using Transwell inserts (Corning, USA). U-87MG cells (1 × 106) were incubated in serum-free medium for 24 h, and the resulting conditioned medium was used as the chemoattractant. MenSC-eGFP cells (1 × 104 cells) were plated in Transwell inserts. Serum-free medium or MEM medium with 30% FBS was placed in the lower wells and used as controls. After 48 hours, the cells on the upper sides of the filters were wiped off, and the cells that had migrated to the lower sides were counted under a fluorescence microscope (IX83, Olympus, Japan).
For the in vivo migration study, the injected cells were analyzed using an IVIS Spectrum 3D Small animal in vivo imaging system (PerkinElmer, USA). Frozen tumor sections were counterstained with4′, 6-diamidino-2-phenylindol (DAPI; Sigma, USA) (1 μg/ml) to detect migration of the MenSC-eGFP and MenSC-sTRAIL cells under a fluorescence microscope.
For the in vivo migration study, the injected cells were analyzed using an IVIS Spectrum 3D Small animal in vivo imaging system (PerkinElmer, USA). Frozen tumor sections were counterstained with4′, 6-diamidino-2-phenylindol (DAPI; Sigma, USA) (1 μg/ml) to detect migration of the MenSC-eGFP and MenSC-sTRAIL cells under a fluorescence microscope.
8
Characterization of Pluripotent Stem Cells
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The alkaline phosphatase activity of PSCs was detected using a kit according to the manufacturer’s instructions (Sigma-Aldrich). Expression of marker proteins of PSCs (LIN28, NANOG, and SSEA1) was analyzed by immunofluorescence staining. The stem cells were grown on cover-slips coated by 0.1% gelatin. The cells were fixed with 4% paraformaldehyde in PBS before blocking in 1% bovine serum albumin solution. For detection of NANOG, the cells were permeabilized (0.1% Triton X-100, Sigma-Aldrich) for 10 min before incubation with the primary antibody (Ab). The primary Abs (Table S1 in Supplementary Material) were added in 100 µl PBS to the cover-slips and incubated for 1 h at 37°C in a humidified chamber. Afterward, the cover-slips were washed three times with PBS before the secondary Ab was added. After incubation for 1 h at 37°C, the cover-slips were washed again before nuclei were stained with 4′,6-diamidino-2-phenylindol (DAPI, 0.4 µg/ml, Sigma-Aldrich) for 10 min. The samples were mounted in Vectashield mounting medium (Vector Laboratories, Cambridgeshire, UK) and analyzed with the fluorescence microscope Axio Observer Z1 (Zeiss, Jena, Germany) and Axio Vision 4.6 software.
9
Microglia Immunohistochemistry in Mouse Brain
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For immunohistochemistry, anti-Iba1 (Abcam ab153696) was used to identify microglia in the brain. Alexa Fluor® 488 AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson 711-545-152) was used. After being anesthetized, the mice were transcardially perfused with PBS and then with 4% paraformaldehyde (PFA) in PBS (wt/vol). Brains were extracted and fixed at 4°C in 4% PFA overnight and then dehydrated in 30% sucrose in PBS. A freezing microtome was used to cut 40-μm coronal slices. The slices were stained with an antibody in a single well. The slices were incubated overnight at 4°C in primary antibodies and then were incubated for 2 h with secondary antibodies at room temperature. For counterstaining, the slices were incubated for 5 minutes with 4’, 6-diamidino-2-phenylindol (DAPI, 0.4 mg/mL, Sigma). All images were obtained with a Zeiss LSM880 confocal microscope.
10
Immunocytochemistry of α-Synuclein and Phosphorylation
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For ICC, cells were washed with PBS and diluted 1:10 Trypsin, fixed with 4% paraformaldehyde (PFA) for 20 min at RT, and followed by a permeabilization step with 0.5% Triton X-100 (Sigma-Aldrich) for 20 min at RT. After blocking in 1.5% normal goat serum (PAA)/DPBS for 1 h, cells were incubated with primary antibody. Primary antibodies used were as follows: anti-aSyn Syn-1 mouse, BD Transduction Laboratories (1:1000); anti-pS129-α-syn Rabbit Abcam (1:1000), incubated overnight at 4 °C and secondary antibody (Alexa Fluor 488 donkey anti-mouse IgG and/or Alexa Fluor 555 goat anti rabbit IgG, (Life Technologies- Invitrogen, Carlsbad, CA, USA) for 2 h at RT. Nuclei were stained with 4′6′-diamidino-2-phenylindol (DAPI, Sigma-Aldrich) (1∶5000 in DPBS) for 10 min. After a final wash, coverslips were mounted by using Mowiol (Sigma-Aldrich) and subjected to fluorescence microscopy. Images were analyzed using LAS AF v.2.2.1 (Leica Microsystems) software.
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