Spreading of cells seeded onto transparent glass coverslips was quantified by phase contrast microscopy on an inverted microscope (Axiovert, Zeiss). Coverslips were pre-marked with a grid template. Adhesion counts were taken at four pre-defined locations on the grid per sample (n=3). Spread cells were defined as those which lacked a refractory halo and were non-spherical in appearance.[33 (link)] Cultures were fixed overnight in 4% paraformaldehyde and blocked with 10% normal goat serum or FBS containing 0.1% v/v Triton X-100 in 1× PBS for 1 hour at room temperature. After rinsing, samples were stained with 6-diamidino-2-phenylindole (DAPI, 2 μg/ml, Roche) to visualize cell nuclei and with rhodamine-conjugated phalloidin (1:200 dilution, Invitrogen) to visualize F-actin. Fluorescent images were obtained with a confocal microscope.
6 diamidino 2 phenylindole dapi
Manufactured by Roche
Sourced in Switzerland
6-diamidino-2-phenylindole (DAPI) is a fluorescent dye used in molecular biology and cell biology applications. It binds strongly to the minor groove of DNA, allowing it to be used as a nuclear counterstain for fluorescence microscopy. DAPI emits blue fluorescence when bound to DNA.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Nacalai Tesque (2 mentions)
Triton x 100, by Nacalai Tesque (2 mentions)
Blocking one, by Nacalai Tesque (2 mentions)
Rhodamine conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Axiovert, by Zeiss (1 mentions)
Donkey anti goat igg, by Biotium (1 mentions)
Cf488a, by Biotium (1 mentions)
Mouse anti oct3 4, by Santa Cruz Biotechnology (1 mentions)
Metamorph image analysis software, by Molecular Devices (1 mentions)
Goat anti sox17, by R&D Systems (1 mentions)
Rabbit anti afp, by Agilent Technologies (1 mentions)
Goat anti alb, by Fortis Life Sciences (1 mentions)
3 protocols using 6 diamidino 2 phenylindole dapi
1
Quantifying Cell Spreading on Glass
2
Immunofluorescence Staining of Hepatocytes
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Cells were fixed with 4% paraformaldehyde (Nacalai Tesque) in phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 (Nacalai Tesque), and blocked with 20% Blocking One (Nacalai Tesque) in PBST (0.1% Tween-20 in PBS). Antibodies were diluted in 20% Blocking One in PBST (0.1% Tween-20 in PBS). Cells were counterstained with 6-diamidino-2-phenylindole (DAPI; Roche Diagnostics). The following antibodies were used: goat anti-Albumin (ALB; A80-129A, 1:200, Bethyl Laboratories), Rabbit anti-Alpha-fetoprotein (Dako, Glostrup, Denmark), Rabbit anti-MRP2 (JA32-01, Invitrogen), Mouse anti-phospho-Histone H3 (Ser10) Antibody, clone 3H10 (05-806, Millipore), Donkey Anti-Goat IgG (H + L) whole antibody CF 568 Dye (20106, Biotium), Donkey Anti-Goat IgG (H + L) CF657 Dye (20048, Biotium), Donkey Anti-Rabbit IgG (H + L) CF488A (20015, Biotium), and Phalloidin-iFluor™ 488 Conjugate anti-AAT (23115, Bioquest).
3
Immunofluorescence Analysis of Hepatic Lineage Markers
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Cells were fixed in 4% paraformaldehyde (Nacalai Tesque) in PBS, permeabilized with 0.1% Triton X-100 (Nacalai Tesque), and were then blocked with 20% Blocking One (Nacalai Tesque, Japan) in PBST (0.1% Tween-20 in PBS). Antibodies were diluted in 20% Blocking One (Nacalai Tesque, Japan) in PBST (0.1% Tween-20 in PBS). Cells were counterstained with 6-diamidino-2-phenylindole (DAPI) (Roche Diagnostics, Switzerland).
The following antibodies were used: rabbit anti-AFP (Dako, Glostrup, Denmark), goat anti-ALB (Bethyl), mouse anti-OCT3/4 (Santa Cruz), goat anti-SOX17 (R&D Systems), and Alexa 568-conjugated and Alexa 488-conjugated antibodies (Invitrogen). Positive cells versus total cells (DAPI-positive cells) were quantified using the Metamorph Image Analysis Software (Molecular Devices).
The following antibodies were used: rabbit anti-AFP (Dako, Glostrup, Denmark), goat anti-ALB (Bethyl), mouse anti-OCT3/4 (Santa Cruz), goat anti-SOX17 (R&D Systems), and Alexa 568-conjugated and Alexa 488-conjugated antibodies (Invitrogen). Positive cells versus total cells (DAPI-positive cells) were quantified using the Metamorph Image Analysis Software (Molecular Devices).
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