Apoptosis was determined using cleaved caspase 3 IF as previously described [12 (link)]. Briefly 40,000 cells were seeded in 12 well plates on glass coverslips in triplicate. 24 hours later cells were treated with chemotherapeutic agents with or without A69 for 24 hours. After treatment, cells were fixed in 3.7% formaldehyde for 15 minutes and permeabilized in 0.3% Triton X-100 for 15 minutes. Antibodies were diluted in blocking buffer that consisted of 0.2% non-fat dry milk, 2% Bovine Serum Albumin and 0.3% Triton X-100 in phosphate buffered saline (PBS). Primary anti-cleaved caspase 3 rabbit monoclonal antibody (1:400, Cell Signaling Technology, Danvers, MA) was applied to cells for 1 hour at 37°C. Following washes in PBS, samples were incubated in goat-anti-rabbit Alexa Fluor 488 (1:1000, Life Technologies, Carlsbad, CA) and Alexa 555 conjugated Phalloidin (1:200, Life Technologies) to visualize F-actin. Slides were mounted with Fluoromount G with Hoescht (Sigma) to label cell nuclei. The percentage of positive cells was determined for each assay with at least 150 cells being counted per condition using the counting feature on an Evos Xl core microscope. Each assay was run in triplicate and repeated three times. Representative images were taken on Zeiss Axio A1 Microscope with an AxioCam MRc digital camera.
Alexa 555 conjugated phalloidin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa 555 conjugated phalloidin is a fluorescent stain used to label filamentous actin (F-actin) in cells. It binds to F-actin and emits red fluorescence when excited by light of the appropriate wavelength, allowing visualization of the actin cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Axio a1 microscope, by Zeiss (2 mentions)
Fluoromount g, by Merck Group (2 mentions)
Axiocam mrc, by Zeiss (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Axiocam mrc digital camera, by Zeiss (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Scmos microscope camera, by Oxford Instruments (1 mentions)
Quanta 450 feg scanning electron microscope, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Te300, by Nikon (1 mentions)
Citrate antigen retrieval buffer, by Agilent Technologies (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Anti zo 1 antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa 647, by Thermo Fisher Scientific (1 mentions)
Lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
Las af software, by Leica (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Alexa 488 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
11 protocols using alexa 555 conjugated phalloidin
1
Quantifying Apoptosis using Cleaved Caspase-3 IF
2
Phospho-H2AX Assay in Cells
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40,000 cells were plated on glass coverslips in a 12 well plate in triplicate and 24 hours later cells were treated as described above. After treatment, cells were fixed in 3.7% formaldehyde for 15 minutes and permeabilized in 0.3% Triton X-100 for 15 minutes. Antibodies were diluted in blocking buffer that consisted of 0.2% non-fat dry milk, 2% Bovine Serum Albumin and 0.3% Triton X-100 in phosphate buffered saline (PBS). Primary anti-phospho H2AX (1:650, Millipore) was applied to cells for 1 hour at 37 °C. Following washes in PBS, samples were incubated in goat-anti-rabbit Alexa Fluor 488 (1:1000, Life Technologies) and Alexa 555 conjugated Phalloidin (1:200, Life Technologies) to visualize F-actin. Slides were mounted with Fluoromount G with Hoescht (Sigma) to label cell nuclei. The percentage of positive cells was determined for each assay with at least 300 cells being counted per condition using the counting feature on an Evos Xl core microscope. Each assay was run in triplicate and repeated three times. Representative images were taken on Zeiss Axio A1 Microscope with an AxioCam MRc digital camera.
3
Optofluidic Channel Imaging Protocol
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FEI Quanta 450 FEG scanning electron microscope (SEM, Dawson, NE) at a scanning voltage of 5 kV and a spot size of 4.0 was applied to capture the images of the optofluidic channel at a tilt angle of 45º.
The cells were stained using immunofluorescence staining method at a floating state.
To stain floating cells, the cells were first resuspended by using 0.25 % trypsin-EDTA in phosphate buffered saline (PBS; Sigma-Aldrich, St. Louis, MO). The cells were fixed with 4 % paraformaldehyde (PFA, Sigma-Aldrich, St. Louis, MO) in phosphate buffered saline (PBS, Sigma-Aldrich, St. Louis, MO) for 10 min and then treated with 0.3% Triton X-100 in PBS for 10 min. We applied 10 % goat serum for 1 hr to avoid non-specific binding of the staining molecules in the next steps. The Lamin-A primary antibody (Abcam, Cambridge, MA) was applied for 1hr. The cytoskeletal actin was stained with Alexa-555 conjugated phalloidin (Life Technologies, Carlsbad, CA) followed by staining the nucleus with 0.1% Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) for 10 min. And the stained images were obtained by using laser confocal microscope (TCS SP8 Confocal Microscope, Leica, Germany). Bright field imaging is conducted by a phase-contrast inverted microscope (TE300, Nikon, Tokyo, Japan) and an sCMOS microscope camera (Zyla, Andor, Belfast, UK) was applied to capture highresolution images (~570 nm/pixel).
The cells were stained using immunofluorescence staining method at a floating state.
To stain floating cells, the cells were first resuspended by using 0.25 % trypsin-EDTA in phosphate buffered saline (PBS; Sigma-Aldrich, St. Louis, MO). The cells were fixed with 4 % paraformaldehyde (PFA, Sigma-Aldrich, St. Louis, MO) in phosphate buffered saline (PBS, Sigma-Aldrich, St. Louis, MO) for 10 min and then treated with 0.3% Triton X-100 in PBS for 10 min. We applied 10 % goat serum for 1 hr to avoid non-specific binding of the staining molecules in the next steps. The Lamin-A primary antibody (Abcam, Cambridge, MA) was applied for 1hr. The cytoskeletal actin was stained with Alexa-555 conjugated phalloidin (Life Technologies, Carlsbad, CA) followed by staining the nucleus with 0.1% Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) for 10 min. And the stained images were obtained by using laser confocal microscope (TCS SP8 Confocal Microscope, Leica, Germany). Bright field imaging is conducted by a phase-contrast inverted microscope (TE300, Nikon, Tokyo, Japan) and an sCMOS microscope camera (Zyla, Andor, Belfast, UK) was applied to capture highresolution images (~570 nm/pixel).
4
Immunolocalization of Tight Junction Proteins
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Sections from the distal small intestine from each treatment group were fixed in 10% formalin and embedded in paraffin wax. A minimum of 10 sections from each of the 5 animals per group were analyzed by immunofluorescence microscopy. Representative images are shown. For immunostaining with Claudin-1 antibody, 8 μm tissue sections were deparaffinized with xylene and tissue was rehydrated through a series of graded alcohol and then processed for antigen retrieval using citrate antigen retrieval buffer (DAKO). Tissue sections were stained with polyclonal rabbit antibody against Claudin-1 (Invitrogen) in PBS with 1% bovine serum albumin (BSA) overnight at 4°C. For immunostaining with anti-ZO-1 antibody, intestinal tissue was frozen in TFM tissue freezing media, and later 5 μm tissue sections were used. Sections were fixed with 1% paraformaldehyde for 10 min at room temperature and incubated with anti-ZO-1 antibody (Invitrogen). After washing, sections were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen); for F-actin staining, sections were incubated with Alexa 555 conjugated phalloidin (Invitrogen) for 1 h at room temperature. Sections were mounted under coverslip using ProLong Gold antifade reagent with DAPI (Invitrogen). Stained sections were imaged using a fluorescence microscope (Leica Microsystems, Germany).
5
Immunofluorescence Analysis of H441 Cells
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H441 cells were grown on Lab-TekII chamberslides (Thermo Fisher Scientific) and treated as indicated. Cultures were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.2% saponin in blocking buffer (10% goat serum, 10 mM Hepes, 10 mM glycine in RPMI 1640) for 15 min at room temperature. Slides were then washed and blocked in blocking buffer for 1 h at room temperature. Indicated antibodies were diluted in blocking buffer incubated with cells at 4 °C overnight. After three washes with PBS, cells were incubated with respective 1:100 diluted secondary antibodies conjugated with either Alexa488 or Alexa647 (Invitrogen). F-actin was detected using Alexa 555 conjugated phalloidin (Invitrogen) diluted 1:40 in blocking buffer. Slides were mounted with Prolong Gold anti-fade reagent containing DAPI and viewed with a LEICA SP5 inverted confocal microscope under a 63 × oil objective, using Leica LAS AF software to acquire images.
6
NMDA-induced Neuronal Morphology Changes
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Rat hippocampal neurons were cultured on poly-L-lysine-coated coverslips. After treatment with NMDA for 4 h, the cells were fixed with 4% (w/v) paraformaldehyde/0.1 M sodium phosphate buffer (pH 7.1) for 1.5 h at 4°C, permeabilized with blocking buffer (0.1 M sodium phosphate buffer containing 0.4% (v/v) Triton X-100, 2% (v/v) donkey serum, and 1% (w/v) bovine serum albumin) for 30 min at room temperature, and then labeled with anti-drebrin A/E (C-term) and anti-NeuN antibodies at 4°C overnight. Alexa 488-conjugated donkey anti-mouse IgG (Invitrogen) and Alexa 647-conjugated donkey anti-rabbit IgG (Invitrogen) were used as secondary antibodies. F-actin was stained with Alexa 555-conjugated phalloidin (Invitrogen). For quantitative analyses of the immunofluorescence data, images of 100 randomly selected NeuN-positive neurons were collected using a BIOREVO BZ-9000 microscope (Keyence, Osaka, Japan) with a 100× oil immersion objective, and the signal intensities of the circular areas (40 μm in diameter) covering both the cell soma and the apical dendrites were measured using Image J analysis software.
7
Visualizing Ovarian Tissue Structure
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We dissected ovaries in phosphate buffered saline (PBS) (pH 7.4) and fixed them in 4% paraformaldehyde in PBS for 30 min at room temperature. After washing them with PBST, we incubated them with a rabbit anti-GFP antibody (1:1000; Invitrogen, A11122) for 48 h at 4°C. After washing, we then incubated the samples for 24 h at 4°C with an Alexa 488-conjugated goat anti-rabbit (1:1000; Invitrogen, A11008) secondary antibody and an Alexa 555-conjugated phalloidin (1:1000; Invitrogen, A34055) to visualize actin in the muscle-rich reproductive tract. Finally, we mounted the tissues in Vectashield and imaged them with an LSM 700/Axiovert 200M confocal microscope (Zeiss). All confocal images are maximal intensity Z-projections. We performed all image processing with ImageJ.
8
Fluorescent Imaging of Skeletal Muscle Tissue
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Muscle tissue isolated from the left hind limb was stuck to a corkboard piece using Tragacanth gum (Wako). Then, the skeletal muscle was rapidly frozen in isopentane (Wako) pre-chilled in liquid nitrogen and stored at −80 °C until sectioning. Seven µm-thick cross-sections were made from muscle samples with a cryostat (Leica, Wetzlar, Germany) at −20 °C, fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS, and then stained with Alexa555-conjugated phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) and Hoechst 33342 (Sigma-Aldrich). After staining, the cross-sections were washed with PBS and mounted using Entellan new (MERCK, Darmstadt, Germany). Stained muscle sections were observed on a fluorescence microscope (BZ-9000: KEYENCE, Osaka, Japan), and captured fluorescent images of the cross-sections were analyzed using the ImageJ software (Fiji package; NIH, Bethesda, MD, USA).
9
Visualizing ASC Speck Formation in NHDF Cells
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NHDF on slides was fixed in PBS-based paraformaldehyde (4%) for 15 min at 25 °C, incubated in Triton X-100 (0.1%) for 10 min, and treated with the blocking solution (normal goat serum) for 1 h at 25 °C. Samples were then stained with primary anti-rabbit ASC antibody overnight and incubated with Alexa 555-conjugated phalloidin (Thermo Fisher Scientific, Hudson, NH, USA) for 2 h. The ASC speck formation was visualized with an Olympus Fluo View™ 300 confocal microscope with a 400 × objective lens.
10
Phalloidin Staining of Actin Cytoskeleton
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For phalloidin
or F-actin staining, the cells were grown on the respective PCL/cellulose
(PCL/TMSC after regeneration)-coated cover slide and washed with PBS
and fixed with 4% formaldehyde for 30 min at 4 °C. Then, the
cells were incubated with Alexa555 conjugated-phalloidin (Molecular
Probes) for 20 min at room temperature followed by repetitive washing
with PBS and mounting with an antifading embedding medium (Vector
Laboratories) containing DAPI counterstaining to visualize the nucleus.
Images (scale bar 20 μm) were obtained by a Zeiss LSM510 META
confocal imaging system with a planeofluar X40/1.3 oil DIC objective
(Jena Germany).
or F-actin staining, the cells were grown on the respective PCL/cellulose
(PCL/TMSC after regeneration)-coated cover slide and washed with PBS
and fixed with 4% formaldehyde for 30 min at 4 °C. Then, the
cells were incubated with Alexa555 conjugated-phalloidin (Molecular
Probes) for 20 min at room temperature followed by repetitive washing
with PBS and mounting with an antifading embedding medium (Vector
Laboratories) containing DAPI counterstaining to visualize the nucleus.
Images (scale bar 20 μm) were obtained by a Zeiss LSM510 META
confocal imaging system with a planeofluar X40/1.3 oil DIC objective
(Jena Germany).
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