Cells were fixed and prepared for immunofluorescence as described previously (Wang et al., 2011 (link)). Coverslips were incubated in primary antibodies for 2 h at 37°C. The primary antibodies used were FLAG (Cell Signaling), MT1-MMP (Abcam), and Tks5 (Santa Cruz). Then the coverslips were washed with d -PBS and incubated in labeled secondary antibodies (Life Technologies) for 1 h at room temperature. Actin was visualized by TRITC-phalloidin or Phalloidin-Atto 390 (Sigma Aldrich) staining. Prolong mounting medium (Life Technologies) was used to mount the coverslips before imaging. Fluorescence images were acquired using epifluorescence microscopes (Axio Observer and Axiovert 200; Carl Zeiss MicroImaging) using a 63× oil objective, unless otherwise indicated, with iVision software. For confocal microscopy, cells were imaged with a Zeiss LSM 780 confocal microscope (Carl Zeiss MicroImaging) and a 63×/1.2-NA water immersion lens. Images were processed and adjusted with Adobe Photoshop software (Adobe) uniformly to the entire image.
Phalloidin atto 390
Manufactured by Merck Group
Phalloidin-Atto 390 is a fluorescent dye used to label and visualize actin filaments in cells. It binds specifically to F-actin, allowing for the detection and analysis of the actin cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Prolong mounting medium, by Thermo Fisher Scientific (2 mentions)
Axio observer, by Zeiss (2 mentions)
Axiovert 200, by Zeiss (2 mentions)
Phalloidin tritc, by Merck Group (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Mt1 mmp, by Abcam (1 mentions)
Labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Flag tag (flag), by Cell Signaling Technology (1 mentions)
Lsm 780 confocal microscope, by Zeiss (1 mentions)
Lsm800 inverted confocal microscope, by Zeiss (1 mentions)
Oregon green conjugated gelatin, by Thermo Fisher Scientific (1 mentions)
Anti mouse irdye 800cw, by LI COR (1 mentions)
Microscope slide, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Eurobio Scientific (1 mentions)
Phalloidin atto 550, by Merck Group (1 mentions)
7 protocols using phalloidin atto 390
1
Immunofluorescence Visualization of Cellular Proteins
2
Sarcomere Visualization in C. elegans
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Sarcomere assembly was monitored by staining F-actin with Phalloidin-Atto 390 (Sigma Aldrich) using the protocol described in (53 ). Briefly, synchronized day 1 adults were washed from NGM plates seeded with E. coli OP50, and the worm pellet was snap-frozen in liquid nitrogen. The pellet was dried in the SpeecVac concentrator, followed by worm permeabilization using 100% ice-cold acetone for 5 min. After acetone removal, the worm pellet was stained using Phalloidin-Atto 390 for 30 min in the dark. Stained worms were washed twice, mounted onto a 2% (w/v) agarose pad on a glass slide, and imaged on Zeiss LSM800 inverted confocal microscope.
3
Gelatin-Coated Coverslip Degradation Assay
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Gelatin-coated coverslips were prepared as described in the presence or absence of Oregon green–conjugated gelatin (Invitrogen), diluted in 0.2% gelatin (Sigma Aldrich; Wang and McNiven, 2012 (link)). Cells were first transfected with the indicated constructs for 1 d or siRNAs for 2 d, then replated onto gelatin-coated coverslips in the presence of the MMP inhibitor BB94 overnight (5 µM; Millipore), and then rinsed at least three times with HBSS and culture medium to provide a synchronized starting point for cell-based gelatin degradation over the indicated time before fixation. The cell border represented by actin staining was visualized by TRITC-phalloidin or Phalloidin-Atto 390 (Sigma Aldrich) and viewed by immunofluorescence. The percentage of cells that degraded the gelatin matrix was scored with ≥100 cells that were randomly imaged. The degradation area of the cells degrading matrix was quantified and normalized to the total cell area with ImageJ software (Martin et al., 2012 (link)).
4
Visualizing Actin Cytoskeleton with Fluorescence Microscopy
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Cells were fixed as described previously [22 (link)]. TRITC-phalloidin and Phalloidin-Atto 390 (Sigma Aldrich) were used to visualize Actin. The coverslips were mounted with Prolong mounting medium (Life Technologies) before imaging. Fluorescence images were acquired using epifluorescence microscopes (Axio Observer and Axiovert 200; Carl Zeiss MicroImaging) using a 63x oil objective with iVision software or Zen software. Adobe Photoshop software (Adobe) was applied to process and adjust the images uniformly.
5
Triobp Mutant Mouse Organ of Corti Explants
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P2–P3 TriobpΔex9–Δex10 mouse organ of Corti explants were kept in culture overnight, and then transfected via Helios gene gun (Belyantseva, 2016 (link)) using 1-µm gold particles coated with DsRed–Triobp-5 cDNA (Katsuno et al., 2019 (link)). They were fixed 24 h later for 40 min at room temperature in 4% formaldehyde (50-980-487; Electron Microscopy Sciences) in 1× PBS and processed for immunostaining using goat or rabbit ANKRD24 antibodies and phalloidin-Atto 390 (50556-10NMOL; Sigma-Aldrich) as described above.
6
Western Blot and Immunofluorescence Antibody Protocol
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The list of primary antibodies is presented in Additional file 1: Table 1.
Secondary antibodies used for Western blotting, diluted 1:10,000, were as follows: horseradish peroxidase (HRP)-conjugated anti-goat, anti-mouse and anti-rabbit (Jackson ImmunoResearch Labs, #805–035-180, #115–035-062, #111–035-144, respectively); secondary fluorophore-conjugated anti-mouse IRDye 800CW and anti-rabbit IRDye 680CW (LI-COR Biosciences, #926–32,212 and #926–68,023, respectively).
For immunofluorescence, the following secondary antibodies were used: Alexa Fluor 488-, 555-, 647-conjugated anti-goat, anti-mouse and anti-rabbit, purchased from Thermo Fisher Scientific, diluted 1:500. Phalloidin-Atto-390 (#50556; 1:1000), used for actin staining, was from Sigma-Aldrich.
Secondary antibodies used for Western blotting, diluted 1:10,000, were as follows: horseradish peroxidase (HRP)-conjugated anti-goat, anti-mouse and anti-rabbit (Jackson ImmunoResearch Labs, #805–035-180, #115–035-062, #111–035-144, respectively); secondary fluorophore-conjugated anti-mouse IRDye 800CW and anti-rabbit IRDye 680CW (LI-COR Biosciences, #926–32,212 and #926–68,023, respectively).
For immunofluorescence, the following secondary antibodies were used: Alexa Fluor 488-, 555-, 647-conjugated anti-goat, anti-mouse and anti-rabbit, purchased from Thermo Fisher Scientific, diluted 1:500. Phalloidin-Atto-390 (#50556; 1:1000), used for actin staining, was from Sigma-Aldrich.
7
Confocal Imaging of Actin Cytoskeleton
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For confocal microscopy experiments, the cells were seeded on glass coverslips and let adhere for 24 hours prior to the double exposure. At the end of the exposure period, the cells were washed twice for 5 min in PBS, fixed in 4% paraformaldehyde for 30 min at room temperature. After two washes (5 min in PBS), they were permeabilized in Triton X100 (0.1% w/v) for 5 min at room temperature. After two more washes in PBS, Phalloidin-Atto 550 (Sigma) (200 nM) or Phalloidin-Atto 390 (500 nM) was added to the cells and let for 20 min at room temperature in the dark. Coverslips-attached cells were washed, and cell nuclei were colored via DAPI for 5 minutes at room temperature, protected from light (1µg/ml, Eurobio, Les Ulis, France). The coverslips were washed twice and placed on microscope slides (Thermo Scientific, Illkirch, France) using a Vectashield mounting medium and imaged using a Zeiss LSM 880 confocal microscope. The images were processed using the ImageJ software.
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