NSC cultures were fixed with 4% Paraformaldehyde (PFA) for 20 min, washed and blocked with 10% Normal Donkey Serum (NDS) in PBS-T for an hour prior to overnight incubation with primary antibody anti-β-Tubulin III (Tuj1, 1:500, Covance, Inc.). Cells were then stained with Alexa 488- or Alexa 546-conjugated secondary antibodies (Invitrogen, Inc., Thermo Fisher) and the nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI) (Sigma).
40 6 diamidino 2 phenylindole dapi
Manufactured by Merck Group
Sourced in United States, France, Germany
40,6-diamidino-2-phenylindole (DAPI) is a fluorescent dye used in molecular biology and microscopy applications. It binds strongly to DNA, allowing for the visualization and identification of cell nuclei. DAPI emits blue fluorescence when bound to DNA, making it a useful tool for nuclear staining and counterstaining.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Observer z1, by Zeiss (2 mentions)
Formaldehyde, by Carl Roth (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Alexa 488 or, by Thermo Fisher Scientific (1 mentions)
Alexa 546 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ab109463, by Abcam (1 mentions)
Triton x 100, by Sangon (1 mentions)
Slowfade gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Ab10530, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Sangon (1 mentions)
Methanol, by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
Alexa fluor 594 affinipure donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Ki67 primary antibody, by Abcam (1 mentions)
Fv1000, by Olympus (1 mentions)
Image pro plus 6, by Olympus (1 mentions)
35 protocols using 40 6 diamidino 2 phenylindole dapi
1
Immunocytochemical Staining of Neuronal Cells
2
Immunofluorescence Analysis of Histone Modifications
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Oocytes or embryos were fixed in phosphate-buffered saline-buffered 4% paraformaldehyde (PFA) and penetrated with 0.5% Triton X-100 (Sangon Biotech, China). They were blocked with 1% bovine serum albumin (BSA) (Sangon Biotech, China) in PBS. They were incubated with primary antibodies diluted in blocking solution. Next, they were washed in PBS, and labeled with secondary antibodies, counterstained with 40,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Finally, they were mounted on glass slides using SlowFade Gold Antifade Reagent (Life Technologies). Imaging was performed on a Zeiss LSM710 confocal microscope. Single frame of each was scanned at the middle focal plane of their nucleus. Semi-quantitative analysis of the fluorescence signals was conducted using the NIH Image program ImageJ. Briefly, the pixel value/unit area was measured for the nucleus, and the value for the cytoplasm was subtracted as background. The value obtained was multiplied by the nuclear area to yield the total amount of fluorescence in the nucleus.
The primary antibodies used were anti-H4K16Ac (Abcam, ab109463, 1:400), anti-B23 (Abcam, ab10530, 1:1000). The secondary antibodies were Alexa Fluor 594-conjugated goat anti-rabbit/mouse IgG (Life Technology) and Alexa Fluor 488-conjugated goat anti-rabbit/mouse IgG (Jackson ImmunoResearch Laboratories, USA).
The primary antibodies used were anti-H4K16Ac (Abcam, ab109463, 1:400), anti-B23 (Abcam, ab10530, 1:1000). The secondary antibodies were Alexa Fluor 594-conjugated goat anti-rabbit/mouse IgG (Life Technology) and Alexa Fluor 488-conjugated goat anti-rabbit/mouse IgG (Jackson ImmunoResearch Laboratories, USA).
3
Fluorescent Staining of Cultured Cells
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A total of 1.2 × 105 cells were grown on poly-L-lysine-coated coverslips for 24 h. Then, cells were treated with 6 , 7 , and 13 , and fixed with methanol (Sigma-Aldrich, Co., Ltd., Gillingham, UK) for 10 min at −20 °C and washed 3 times with PBS for 5 min. DNA was stained with 2 µg/mL 40,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) diluted in Vectashield mounting medium (Vector, H-1000, Burlingame, CA, USA).
4
Detecting Ki67 in Aging BMSCs
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Expression of Ki67 was detected by cell immunofluorescence in aging-BMSCs treated with or without apocynin. After harvest, cells were fixed in 4% paraformaldehyde and then treated with 0.5% triton X-100 (Sigma-Aldrich, US) for 20 min. After blocking with 5% goat serum (Millipore, US), samples were incubated in Ki67 primary antibody (Abcam, US) overnight at 4 °C. On the next day, samples were treated with Alexa Fluor 594 affinipure donkey anti-rabbit IgG (Jackson, US) and the 0.1 mg/ml of 40, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, US) was used to stain the nuclear. All samples were observed under a fluorescence microscope (FV1000, Olympus, Japan) and the average optical density was calculated by Image Pro Plus 6.0.
5
Immunofluorescent Osteocalcin Staining
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The cells were washed twice with PBS and fixed on a glass slide with 4% formalin (ScyTek Laboratories, Logan, UT) for 30 min at room temperature (RT), then washed with PBS, and permeabilized/blocked with 0.3%Triton X-100, goat serum 5%, and 1% BSA in PBS for 45 min at RT. After adding blocking solution, the washed cells were incubated with the primary antibody mouse anti-osteocalcin (1 : 100 R&D System 962643) overnight at 4°C. Subsequently, the cells were incubated for 1 hour in the dark at RT with the secondary antibody anti-mouse Alexafluor 488 (1 : 1000) (Invitrogen, Life Technologies Corporation, NY) and with 1 μg/ml of 40,6-diamidino-2-phenylindole (DAPI, Sigma) to stain cells nuclei.
6
Immunolocalization of Phospho-GSK3α in Boar Sperm
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Boar sperm were fixed in 4% neutral paraformaldehyde for 30 min, treated with TBS containing 0.5% Triton X-100 at room temperature for 1 h, blocked with 10% FCS and then incubated with rabbit anti-Phospho-GSK3α (Ser21) antibody (Cell Signaling Technology, 9316, MA, USA) overnight at 4°C. The tetramethylrhodamine-labeled goat anti-rabbit IgG (1:1.000, KPL Inc., Gaithersburg, MD, USA) were used as the second antibody. The 40, 6-diamidino-2-phenylindole (DAPI, Sigma Aldrich, MO, USA) was used as a marker for cell nuclei. Negative control sections were incubated with normal serum instead of primary antibodies. Mounted slides were visualized using a fluorescence microscope (Leica, DMI6000 B, Germany).
7
Immunofluorescence Staining of Neurons
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Cells and DRGs were fixed at different times post-seeding in 4% paraformaldehyde solution in PBS for 15 min and 45 min respectively at room temperature and rinsed three times with PBS. Samples were permeabilized in 0.5% Triton X-100 in PBS for 5 min at room temperature and rinsed twice with PBS before 1 h incubation at room temperature in 1% bovine serum albumin (BSA) in PBS. Rabbit primary antibody directed against βIII tubulin (Sigma-Aldrich, France) was diluted (1∶150) in PBS/BSA 0.1% (w/v) and incubated at room temperature for 1 h. The cells were washed two times with 0.1% BSA in PBS. Cy3-conjugated secondary antibody (cy3 goat anti-rabbit IgG, Jackson Immunoresearch), 40,6-diamidino-2- phenylindole (DAPI; 1 µg.mL−1, Sigma) and Phalloidin-X5- 505 (0.16 nmol ml−1, Fluoroprobes, France) were added and incubated at room temperature in the dark for 1 h. The samples were then rinsed with PBS, mounted in Mowiol and observed using a LEICA DMI 6000 microscope or a LSM 710 scanning confocal microscope which images were analyzed with ‘Zen 2011’ software.
8
Immunofluorescent Detection of Apoptosis Markers
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Samples were first washed with PBS and fixed in 4% formaldehyde (Carl Roth, Karlsruhe, Germany) for 10 min (2D) or 15 min (3D) at room temperature. After washing twice with PBS, the samples were treated with 1% bovine serum albumin (BSA) blocking solution containing 0.1% (v/v) Triton X-100 (Carl Roth) for 1 h. Afterward, the blocking solution was discarded, and samples were incubated with the respective primary antibody (anti-cleaved PARP (Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb #5625, 1:400, Cell Signaling Technology, Danvers, MA, USA; anti-cleaved caspase3, 1:400, Cell Signaling Technology) overnight at 4 °C. The samples were then washed three times with PBS and incubated with the corresponding secondary antibodies (goat anti-mouse Alexa Fluor 594, 1:500, Invitrogen, Carlsbad, CA, USA; goat anti-rabbit Alexa Fluor 488, 1: 500, Invitrogen) for 1 h at room temperature. After washing twice with PBS, nuclear staining was performed with 1 μg/mL of 40,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) for 30 min, and samples were washed once again with PBS. Stained samples were analyzed by fluorescence microscopy (Observer Z1, Carl Zeiss).
9
Immunofluorescence Imaging of Transfected Mesangial Cells
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Glomerular mesangial cells were seeded on the glass cover slips. After transfection with plasmids or siRNA targeting PAQR3 for 24 h, the cells were washed with cold phosphate-buffered saline, fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% TritonX-100 for 10 min, blocked with 10% goat serum for 1 h, and then incubated with primary antibodies overnight at 4°C. After washing, the cells were incubated with fluorescent secondary antibody in the darkroom at room temperature for 1 h. The nuclei were labeled with 40, 6-diamidino-2-phenylindole (DAPI, Sigma, USA) for 10 min. Finally, the images were captured using a laser scanning confocal fluorescence microscope (LSM510, Carl Zeiss, Germany).
10
Immunofluorescence Imaging of FAK and HER2
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SKBR3 and BT-474 cells were grown on coverslips previously coated with 1% sterile gelatin (Sigma-Aldrich) and exposed for 72 h to 1-10 μg/ml Tz, 10-6M RA or the combination of both drugs. Cells were fixed with 4% paraformaldehyde for 35 min and permeabilized with 0.1% Triton for 5 min. Blocking step was performed with 0.5% bovine serum albumin solution for 30 min at room temperature. Cells were incubated with the first antibody against FAK (Mouse, clone 77, BD Biosciences) and against HER2 (Mouse, catalogue 16901-Abcam) overnight at 4°C. After washing, cells were incubated with Goat Anti-Mouse IgG-Alexa Fluor 488 (A-11001, Invitrogen) for 90 min at room temperature. The cells were washed and then stained for 35 min with Texas Red phalloidin (Sigma-Aldrich) to reveal actin and the nuclei counter stained with 40-6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 10 min. The coverslip cells were mounted with Vectashield mounting media (Vector Laboratories, Burlingame, CA, USA). Immunofluorescence images were captured by using a Nikon Eclipse E200 microscope (Tokyo, Japan) coupled to a high-resolution 590CU 5.0M CCD digital camera or examined under fluorescence microscopy (FV1000 Olympus Confocal Microscope) and the FV 10-ASW 1.7 software (Olympus, Japan).
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