Cells were fixed in 4% (v/v) formaldehyde for 7 min, washed twice in PBS containing 1% BSA, and permeabilized with 0.1% (v/v) Triton-X 100 for 1 h. The samples were incubated overnight in ice with primary antibody against anti-sarcomeric α-actinin (Clone EA-53, 1:300, Sigma) followed by 45 min incubation in RT with Alexa 488-conjugated donkey anti-mouse antibody (715-545-151, 1:250, Jackson) and Alexa-Fluor 546-conjugated phalloidin (A22283, 1:250, Life Technologies) which was used for staining F-actin. Before analysis the samples were washed twice with PBS containing 1% BSA and NucBlue (R37605, Invitrogen) was added for nuclei detection. Imaging was performed with Nikon C1si laser scanning confocal microscope (LSCM). Sarcomeric striation of single cells was quantified with the ImageJ software using the OrientationJ plugin and the distribution application [http://big.www.epfl.ch/demo/orientation/ ]. OrientationJ calculates the distribution of orientations of fibers in an image by evaluating the structure tensor39 (link). The extent of striation was determined as the percentage of actinin fibers that are aligned within 5° of the direction of the alignment which was determined as the mode of the angle distribution (Fig. S1.3 ).
Alexa fluor 546 conjugated phalloidin
Manufactured by Thermo Fisher Scientific
Sourced in France, United States
Alexa Fluor 546-conjugated phalloidin is a fluorescent dye-labeled molecule used to visualize F-actin in fixed cells. It binds specifically to actin filaments, allowing for the detection and localization of the cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Objective lens, by Leica (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
N cadherin, by BD (3 mentions)
Alexa fluor 488 conjugated goat anti mouse, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Dm4000b, by Leica (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Nucblue, by Thermo Fisher Scientific (2 mentions)
Tcs sp2 aobs, by Leica (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Polyclonal goat anti osteocalcin, by Santa Cruz Biotechnology (2 mentions)
Monoclonal mouse anti bspii, by Santa Cruz Biotechnology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Goat serum, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Hoechst 33258, by Merck Group (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Lsm 510, by Zeiss (2 mentions)
42 protocols using alexa fluor 546 conjugated phalloidin
1
Quantifying Sarcomeric Striation in Single Cells
2
Immunofluorescence Staining of HCECs
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HCECs cultured on a 48-well cell culture plate were fixed in 0.5% paraformaldehyde for 45 minutes, permeabilized with 1%Triton X-100 for 5 minutes, and incubated with 2% bovine serum albumin (BSA) for 30 minutes. The HCECs were incubated with the following primary antibodies for 45 minutes at 37°C: ZO-1 (1:200, Zymed Laboratories, South San Francisco, CA), N-cadherin (1: 300, BD Biosciences), and Na+/K+-ATPase (1:200, Merck Millipore). Either Alexa Fluor 488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific) or Alexa Fluor 594-conjugated goat anti-mouse IgG (Thermo Fisher Scientific) was diluted at 1:500 in BSA, and the samples were incubated in the secondary antibodies for 45 minutes at 37°C. Actin was stained by incubating samples with a 1:200 diluted Alexa Fluor 546-conjugated phalloidin (Life Technologies Corp.) for 45 minutes at 37°C. Nuclei were stained with 1:1000 diluted 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Vector Laboratories, Burlingame, CA). The samples were examined by fluorescence microscopy (BZ-9000; Keyence, Osaka, Japan).
3
3D Bioprinted Construct Immunofluorescence
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For immunofluorescence of 3D bio-printed structures, constructs were fixed in 4% (v/v) warm methanol free formaldehyde in DMEM buffer (1.8 mM CaCl2·H2O, 5.36 mM KCl, 0.81 mM MgSO4·7H2O, 0.1 M NaCl, 0.44 mM NaHCO3 and 0.9 Mm NaH2PO4) pH 7.4 for 20 min, washed 3 times in DMEM buffer, and permeabilized using Triton-X 100 (0.2% v/v in DMEM buffer) for 15 min at RT. The samples were washed 3 times, blocked for 1 h at RT in DMEM containing 1% BSA. The samples were incubated overnight with the primary antibodies, followed by 2-h incubation with secondary antibodies, with 3 × 10 min PBS washing between.
Alexa-Fluor 546-conjugated phalloidin (A22283, 1:1000, Life Technologies) was used for staining F-actin and NucBlue (Invitrogen) for nuclei detection. Imaging was performed with a Nikon C1si laser-scanning confocal microscope (LSCM). For reconstruction of 3D constructs, images were taken in 150 µm intervals between planes.
For quantification of cells 3D organization, cells perimeters were identified and the aspect ratio of each region of interest (ROI) was calculated based on F-actin staining, using imageJ thresholding and particle analysis of ROIs larger than 200 µm2.
Alexa-Fluor 546-conjugated phalloidin (A22283, 1:1000, Life Technologies) was used for staining F-actin and NucBlue (Invitrogen) for nuclei detection. Imaging was performed with a Nikon C1si laser-scanning confocal microscope (LSCM). For reconstruction of 3D constructs, images were taken in 150 µm intervals between planes.
For quantification of cells 3D organization, cells perimeters were identified and the aspect ratio of each region of interest (ROI) was calculated based on F-actin staining, using imageJ thresholding and particle analysis of ROIs larger than 200 µm2.
4
Immunohistochemical Analysis of Rabbit Corneas
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Corneas were fixed in 4% buffered paraformaldehyde and processed for paraffin embedding. Sections cut 5 μm thick were stained with PAS according to standard protocols. Rabbit corneal specimens were fixed in 4% formaldehyde and incubated for 30 minutes in 1% bovine serum albumin (BSA) to block nonspecific binding. Corneas were examined by actin staining performed with a 1:400 dilution of Alexa Fluor® 546-conjugated phalloidin (Life Technologies Corp.). For immunohistochemical analyses, specimens were incubated overnight at 4°C with primary antibodies against Na+/K+-ATPase (1:300, Upstate Biotechnology, Lake Placid, NY), ZO-1 (1:300, Life Technologies Corp.), and N-cadherin (1:300, BD Biosciences, San Jose, CA). The specimens were then incubated for 2 hours at 4°C with secondary antibody, Alexa Fluor® 488-conjugated goat anti-mouse (1:1000, Life Technologies Corp.). Nuclei were stained with DAPI (Vector Laboratories, Burlingame, CA). The slides were examined with a fluorescence microscope (TCS SP2 AOBS; Leica Microsystems, Wetzlar, Germany).
5
Immunofluorescence Staining of DNA Damage
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About 1.0 × 104 of HeLa cells in 200 µl culture medium were seeded on a glass slide (8-well LabTek II Chamber slide system; Nalge
Nunc, Rochester, NY, U.S.A.) and cultured with 10 µl of filter sterilized bacterial sonic cell lysate at 37°C for 24 hr under 5% CO2in air. Subsequently, cells were fixed with 3.7% formaldehyde in PBS for 10 min, followed by treatment with 0.5% Triton X-100 for 20 min and 1.0% BSA in PBS for
1 hr at room temperature. γH2AX in HeLa cells was stained with anti-phospho-histone-H2AX (Ser139) polyclonal antibody (Enzo Life Sciences, Inc., Farmingdale,
NY, U.S.A.) at 37°C for 1 hr and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Life Technologies, Carlsbad, CA, U.S.A.) at 37°C for 1 hr under dark
condition, respectively. Intracellular F-actin was stained with Alexa Fluor 546-conjugated phalloidin (Life Technologies) at 37°C for 1 hr under dark condition.
Fluorescence was observed under an epifluorescence DM2500 microscope (Leica Microsystems, Wetzlar, Germany).
Nunc, Rochester, NY, U.S.A.) and cultured with 10 µl of filter sterilized bacterial sonic cell lysate at 37°C for 24 hr under 5% CO2in air. Subsequently, cells were fixed with 3.7% formaldehyde in PBS for 10 min, followed by treatment with 0.5% Triton X-100 for 20 min and 1.0% BSA in PBS for
1 hr at room temperature. γH2AX in HeLa cells was stained with anti-phospho-histone-H2AX (Ser139) polyclonal antibody (Enzo Life Sciences, Inc., Farmingdale,
NY, U.S.A.) at 37°C for 1 hr and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Life Technologies, Carlsbad, CA, U.S.A.) at 37°C for 1 hr under dark
condition, respectively. Intracellular F-actin was stained with Alexa Fluor 546-conjugated phalloidin (Life Technologies) at 37°C for 1 hr under dark condition.
Fluorescence was observed under an epifluorescence DM2500 microscope (Leica Microsystems, Wetzlar, Germany).
6
Immunostaining of Rabbit Corneal Epithelial Cells
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Rabbit corneal specimens were fixed in 4% formaldehyde and incubated for 30 minutes in 1% bovine serum albumin (BSA) to block nonspecific binding. Corneas were examined by actin staining performed with a 1:400 dilution of Alexa Fluor® 546 conjugated Phalloidin (Life Technologies Corp.). For immunohistochemical analyses, specimens were incubated with primary antibodies against Na+/K+-ATPase (1:300, Upstate Biotechnology, Lake Placid, NY), ZO-1 (1:300, Life Technologies Corp.), and N-cadherin (1:300, BD Biosciences, San Jose, CA), and then Alexa Fluor® 488-conjugated goat anti-mouse (Life Technologies Corp.) was used as a secondary antibody at a 1:1000 dilution. Proliferative cells were evaluated by 5-ethynyl-2 Click-iT® EdU imaging kits (Life Technologies Corp.) according to the manufacturer’s instructions. Briefly, the RCECs (1 × 104 cells/well) were cultured in a 96-well plate and incubated with 10 μM EdU for 6 h at 37 °C. Following fixation with 4% paraformaldehyde and permeabilization with 0.3% Triton® X-100 (Nacalai Tesque, Kyoto, Japan), the RCECs were incubated with a reaction cocktail for 30 min at room temperature. Nuclei were stained with DAPI (Vector Laboratories, Burlingame, CA). The slides were examined with a fluorescence microscope (TCS SP2 AOBS; Leica Microsystems, Wetzlar, Germany).
7
Immunofluorescence Assay for Myc-tagged Proteins
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Hek293 and HeLa cells were grown in an 8-well slide (ibidi 80826) in DMEM with 10% FBS (Biological Industries, Beit Haemek, Israel). Cells were fixed with 1:1 ethanol methanol solution at −20 °C and placed in 1% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS for 1 h to block non-specific binding. The samples were incubated at room temperature for two hours with an anti-myc (present from Prof. Noah Isakov) antibody. Cells were stained with Alexa-Fluor 546-conjugated phalloidin (A22283; Life Technologies) and Cy2 conjugated anti-c-myc (ZRB1003; Sigma-Aldrich) for 1 h at room temperature, followed by three washes with PBS ×1 (5 min for each). Nuclei were visualized by adding Hoechst 33342 (H3570; Life Technologies). Images were acquired with the confocal FluoView 1000 fluorescence microscope (OLYMPUS), with a 60× objective and the manufacturer’s software FV1000.
8
Quantifying Keratinocyte Morphology Changes
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Keratinocytes were seeded and treated as described above for a period of 2 or 4 days. At the termination of the experiment, cells were rinsed with PBS and fixed with freshly prepared 4% (w/v) paraformaldehyde for 20 min. Cells were permeabilized with 0.1% Triton X-100 for 20 min, blocked with 3% bovine serum albumin for 1 h, and stained with 1 U Alexa Fluor® 546 conjugated-phalloidin (Life Technologies) for 30 min. Images were taken using an Olympus IX51 coupled with Hamamatsu digital camera at 20x magnification. Cell area of individual cells in the bulk (100+ cells from the wound edge) and at the wound edge (<3 cells from the edge) were quantified by ImageJ software.
9
Assessing CD33-Mediated F-Actin Induction
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Example 16
The purpose of this Example is to test whether antagonistic anti-CD33, or CD33 bispecific antibodies induce F-actin in microglial cells, macrophages, and dendritic cells.
Microglia, macrophages or dendritic cells and other cells of interest that are transduced with CD33 or that express CD33 are added to culture plates and then exposed to antagonistic anti-CD33 and/or CD33 bispecific antibodies, or a control antibody. Cells are fixed, blocked, and then stained with Alexa Fluor 546-conjugated phalloidin (Molecular Probes) after 1 h and F-actin is labeled with a fluorescence dye. Images are collected by confocal laser scanning microscopy with a 40× objective lens (Leica). (JEM (2005), 201, 647-657).
10
Siglec-9 Antibody Effects on Immune Cells
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Example 16
The purpose of this Example is to test whether antagonistic anti-Siglec-9 antibodies, or Siglec-9 bispecific antibodies induce F-actin in microglial cells, macrophages, neutrophils, NK cells, and dendritic cells.
Microglia, macrophages, neutrophils, NK cells, or dendritic cells and other cells of interest that are transduced with Siglec-9 or that express Siglec-9 are added to culture plates and then exposed to antagonistic anti-Siglec-9 and/or Siglec-9 bispecific antibodies, or a control antibody. Cells are fixed, blocked, and then stained with Alexa Fluor 546-conjugated phalloidin (Molecular Probes) after 1 h and F-actin is labeled with a fluorescence dye. Images are collected by confocal laser scanning microscopy with a 40× objective lens (Leica). (JEM (2005), 201, 647-657).
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