Total, cytoplasmic and nuclear proteins were prepared using ReadyPrep Protein Extraction Kit (Bio-Rad, Hercules, CA) and aliquots of them were used for western blot analysis with antibodies to IκBβ, RELA, H3 and β-actin (Abcam, Cambridge, MA). The immunoreactive bands were detected by ECL kit and visualized with ImageQuant LAS 4000. The collected culture medium was used for the assay of ICAM1, VCAM1, SELE, SELP and IL-6 by ELISA kits (Abcam, Inc.). HUVECs were cultured on slides in 6-wells plates and transfected with let-7e mimic and negative control as above mentioned. Then, cell immunofluorescence assay was performed using anti-NF-κB mouse monoclonal primary antibody (Cell Signaling Technology, USA) and Alexa Fluor 594-labelled goat anti-mouse IgG secondary antibody (Jackson, US) as described previously50 . DAPI (6-diamidino-2- phenylindole, Sigma-Aldrich, USA) was used to stain the nuclear. The slides were observed under LSM 710 laser confocal microscope and analyzed using ZEN software (Zeiss, Germany).
6 diamidino 2 phenylindole dapi
Manufactured by Merck Group
Sourced in United States, Germany
6-diamidino-2-phenylindole (DAPI) is a fluorescent dye used in molecular biology and microscopy applications. It binds strongly to DNA, specifically to adenine-thymine (A-T) rich regions. DAPI can be used to stain and visualize cell nuclei in a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (3 mentions)
Axioimager microscope, by Zeiss (3 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Ix81 spinning disk confocal microscope, by Olympus (2 mentions)
Mouse anti map2, by Merck Group (2 mentions)
Slowface light antifade reagent, by Thermo Fisher Scientific (2 mentions)
Lsm 710 laser confocal microscope, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Readyprep protein extraction kit, by Bio-Rad (1 mentions)
β actin, by Abcam (1 mentions)
Alexa fluor 568 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
T6793, by Merck Group (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Neutral buffered formalin, by Merck Group (1 mentions)
Xds 3fl4, by Optika (1 mentions)
Anti skp1, by Cell Signaling Technology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscopy, by Zeiss (1 mentions)
A1r mp, by Nikon (1 mentions)
Alexa fluor 488 labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
43 protocols using 6 diamidino 2 phenylindole dapi
1
Protein Extraction and Immunoassay Protocol
2
Immunofluorescence Staining for Ki67 and Cilia
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Deparaffinized sections or fixed cells were permeabilized, blocked, and incubated with primary anti-Ki67 antibody (1:50 for slides, 1:200 for cells, ab16667) overnight at 4 °C. The slides were washed and stained with Alexa Fluor 568-conjugated anti-rabbit IgG (1:1000, Invitrogen) antibody or for 1 h at room temperature. DAPI (6-diamidino-2-phenylindole, Sigma) was used as a counterstain for nuclei .
The same staining procedure was used for acetylated α-tubulin (1:500, T6793, Sigma). For primary cilia staining of DPSCs, the cells were serum starved for 48 h to induce ciliogenesis.
The same staining procedure was used for acetylated α-tubulin (1:500, T6793, Sigma). For primary cilia staining of DPSCs, the cells were serum starved for 48 h to induce ciliogenesis.
3
Assessing Tight Junctions in ARPE-19 Cells
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Immunofluorescence against zonula occludens-1 (ZO-1) was performed to assess the effect of both N and ND treatments on intercellular tight junctions. ARPE-19 cells were seeded at a density of 100,000 cells per well on laminin-coated polycarbonate membrane cell culture inserts (Corning Life Science, Tewksbury, MA, USA) and were grown in 1% serum-free DMEM for 4 weeks. Immunofluorescence was then performed using a ZO-1 anti-rabbit Alexa Fluor 594 antibody (1:100, 339194, Invitrogen- Life Technologies, Carlsbad, CA, USA) diluted in blocking buffer following the same protocol as previously described [36 (link)]. DAPI (6-diamidino-2-phenylindole; Sigma-Aldrich) was used to stain cell nuclei. Images were obtained with a laser scanning confocal imaging system (LSM800, Zeiss, Oberkochen, Germany). N and ND treatments (62.34 µg/mL) were added individually to the cell line in order to be compared to the saline group, and were also added with either H2O2 (1600 µM) or LPS (as described in Section 2.3 ) in order to identify potential recovery effects on cell integrity.
4
Quantifying Decellularization Efficiency
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To characterize decellularization rate, decellularized and untreated sections were fixed in 10% neutral buffered formalin (Sigma, USA). For dehydration, they were passed through increasing concentrations of ethanol (50%, 70%, 90%,100%) for 35 minutes, immersed twice in xylene for 35 minutes, and were then stained using hematoxylin & eosin (H&E) to determine the rate of decellularization, and after that the number of removed nuclei was calculated. For further confirmation of remaining nuclei in decellularized scaffolds, 6-diamidino-2-phenylindole (DAPI) staining (Sigma-Aldrich, USA) was used and examined using fluorescence microscope (OPTIKA- XDS-3FL4, Italy, X10 and 40) by applying applicable filters. To evaluate the collagen status in the decellularized tissue, the section from both control and treated groups was stained with Masson’s trichrome.
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To characterize decellularization rate, decellularized and untreated sections were fixed in 10% neutral buffered formalin (Sigma, USA). For dehydration, they were passed through increasing concentrations of ethanol (50%, 70%, 90%,100%) for 35 minutes, immersed twice in xylene for 35 minutes, and were then stained using hematoxylin & eosin (H&E) to determine the rate of decellularization, and after that the number of removed nuclei was calculated. For further confirmation of remaining nuclei in decellularized scaffolds, 6-diamidino-2-phenylindole (DAPI) staining (Sigma-Aldrich, USA) was used and examined using fluorescence microscope (OPTIKA- XDS-3FL4, Italy, X10 and 40) by applying applicable filters. To evaluate the collagen status in the decellularized tissue, the section from both control and treated groups was stained with Masson’s trichrome.
5
Antibody Validation for Cellular Analysis
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Anti-p53 antibodies (cat. no. sc-126) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-α-SMA (cat. no. 14968), anti-vimentin (cat. no. 5741), anti-Smad2/3 (cat. no. 8685s), anti-p-Smad2/3 (cat. no. 8828), and anti-Skp1 (cat. no. 2156) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). Anti-β-actin (cat. no. A5316) antibodies and 6-diamidino-2-phenylindole (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO, USA).
6
RGNNV Infection in GS Cells
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Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction as previously described (Huang et al., 2009 (link)). Briefly, GS cells were seeded into 24-well plates for 18 h, after which the cells were transfected with a mixture of Lipofectamine 2000 and pEGFP-N3, pEGFP-LC3, pEGFP-Rab7, or pEGFP-Rab5, respectively. After 24 h from the time of transfection, cells were infected with RGNNV for another 24 h, and then fixed in 4% paraformaldehyde for 1 h at 4°C. Finally, cells were stained with 1 μg/mL of 6-diamidino-2-phenyl-indole (DAPI, Sigma), and then observed under fluorescence microscopy (Zeiss).
7
Immunofluorescence of FLAG-NS1 and Myc-STAU2
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Chicken DF1 cells transiently transfected with FLAG-NS1 and Myc-STAU2 plasmids were cultured for 24 h and were then fixed in 4% paraformaldehyde for 30 min at room temperature and were permeabilized with 0.1% Triton X-100 (Sigma, # T8787) for 15 min. FLAG-NS1 cells were incubated with an anti-FLAG antibody (Abmart, # M20008L) and Myc-STAU2 cells with an anti-Myc antibody (Abmart, # M20002L), both at a 1:1,000 dilution for 2 h. After three washes in PBS, the cells were subsequently incubated with a FITC-conjugated secondary antibody (Abcam, # ab6785) for FLAG and with a Cy3.5-conjugated secondary antibody (Abcam, # ab6954) for Myc, both at a 1:1,000 dilution for 1 h. The nuclei were stained with 6-diamidino-2-phenylindole (DAPI) (Sigma, # D9542, 1:500). Finally, the cells were visualized with a confocal microscope (Nikon A1 R MP).
8
Quantifying EGFR Nuclear Translocation
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Cells were plated on 12 mm ø glass coverslips, starved overnight and treated according to the experimental design. Cells were fixed and incubated with anti-EGFR antibody followed by AlexaFluor® 488-labeled secondary antibody (Invitrogen, Carlsbad, CA, USA) as previously described [78 (link)]. 6-diamidino-2- phenylindole (DAPI) 1 μg/ml (Sigma-Aldrich) was used to stain the nuclei. Cells were imaged with a confocal laser scanning microscope Leica SP5. Images for documenting EGFR nuclear translocation were acquired in the middle section of the nuclei with 63× magnification. Confocal stacks were 3D-reconstructed with Imaris Software (Bitplane, Zurich, Switzerland).
9
Autophagy Markers in Neurodegeneration
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Immunofluorescence was used to evaluate the distribution and expression of LC3-II, Beclin1, and P62 in SH-SY5Y cells from normal control and OGD/R groups. For this purpose, the cells were incubated with antibodies against LC3 (1:800; Novus; NB600-1384), Beclin1 (1:100; Abcam Cat# ab55878) and P62 (1:100; Abcam Cat# ab91526), respectively, in a humidified container at 4°C for 12 hr. The cells were rinsed in PBS for 3 times, and incubated with TRITC conjugated anti-rabbit IgG (1:100, Proteintech) at room temperature for 4 hr. 4, 6-diamidino-2-phenylindole (DAPI, 0.0001%, Sigma) was applied to stain nuclei. The cells were examined by a laser confocal microscope (Nikon D-Eclipse C1, Japan).
10
Characterization of Germ Cell Subpopulations
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The identity of SSCs, spermatocytes and spermlike cells was verified by tracking the promyelocytic
leukaemia zinc finger protein (PLZF), synaptonemal
complex protein 3 (SCP3) and acrosin binding protein
(ACRBP) (22 (link)-24 (link)). These markers were detected after 8
weeks of culture. For immunohistochemistry, primary
antibody, mouse monoclonal anti-mouse antibody
against PLZF, SCP3 or ACRBP (1:100, Santa Cruz
Biotechnology, Germany) was added and the samples
were incubated at 4°C overnight. The secondary antibody
Alexa 488-conjugated anti-mouse IgG (1:200, Sigma,
Germany) was added for 2 hours at 37°C in the dark. For
nuclear staining 4′,6-diamidino-2-phenylindole (DAPI,
1:200, Sigma, Germany) was applied for 1 minute. The
specimens were observed with a fluorescence microscope
(Olympus, type CH2, Japan). To quantify the results,
germ cells were defined as cells that stained positive for
PLZF, SCP3 and ACRBP. The results are reported as the
percentage of germ cells that were positive for the protein
of interest relative to the entire population. From each
sample, 5 sections were randomly selected and after highmagnification photography (magnification: x400), 5 fields
from each section were analyzed by image-j software.
leukaemia zinc finger protein (PLZF), synaptonemal
complex protein 3 (SCP3) and acrosin binding protein
(ACRBP) (22 (link)-24 (link)). These markers were detected after 8
weeks of culture. For immunohistochemistry, primary
antibody, mouse monoclonal anti-mouse antibody
against PLZF, SCP3 or ACRBP (1:100, Santa Cruz
Biotechnology, Germany) was added and the samples
were incubated at 4°C overnight. The secondary antibody
Alexa 488-conjugated anti-mouse IgG (1:200, Sigma,
Germany) was added for 2 hours at 37°C in the dark. For
nuclear staining 4′,6-diamidino-2-phenylindole (DAPI,
1:200, Sigma, Germany) was applied for 1 minute. The
specimens were observed with a fluorescence microscope
(Olympus, type CH2, Japan). To quantify the results,
germ cells were defined as cells that stained positive for
PLZF, SCP3 and ACRBP. The results are reported as the
percentage of germ cells that were positive for the protein
of interest relative to the entire population. From each
sample, 5 sections were randomly selected and after highmagnification photography (magnification: x400), 5 fields
from each section were analyzed by image-j software.
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