Alexa 555

Manufactured by Thermo Fisher Scientific

Sourced in United States, France, Panama, Germany, United Kingdom, Colombia

Alexa 555 is a fluorescent dye used in various biological applications. It can be used for labeling proteins, nucleic acids, and other biomolecules. The dye has an absorption maximum at 555 nm and an emission maximum at 565 nm, making it suitable for detection and imaging with common fluorescence techniques.

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299 protocols using alexa 555

Preparation of Alexa 555-labeled Streptavidin

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Atto655 labeled streptavidin was purchased from AttoTec GmbH (Siegen, Germany). Alexa 555 labeled SA was prepared in our own lab as follows: Streptavidin was labeled via N-hydroxysuccinimid (NHS)-ester conjugate Alexa555 (Lifetechnologies, Vienna, Austria) according to protocols provided by AttoTec GmbH (Siegen, Germany). For the labeling, 30 μl of a 1 mg∕ml streptavidin (dissolved in PBS buffer) stock solution were adjusted to a pH of 8.6 with a 0.2-M sodium bicarbonate buffer. A labeling ratio of 1:3 streptavidin to fluorophore has been adjusted. The reaction mixture was incubated for 1 h at room temperature. The labeling solution was purified via a PD-10 Sephadex® G-25 m (GE Healthcare, Pittsburgh) purification column in PBS buffer. To concentrate the labeled streptavidin, the solution was transferred to a cut off filter (3–10.000 MWCO (Molecular Weight Cut Off) PES) and centrifuged at 1200 rpm in a centrifuge (Thermo, Multifuge 1S-R, HERAEUS, Thermo Scientific, Vienna, Austria) until only 100–200 μl remained. The concentrated solution was split into aliquots and stored at −20°C.

Wolfesberger C., Wollhofen R., Buchegger B., Jacak J, & Klar T.A. (2015). Streptavidin functionalized polymer nanodots fabricated by visible light lithography. Journal of Nanobiotechnology, 13, 27.

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Immunocytochemistry of Neural Cell Types

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For immunocytochemistry, samples were plated onto poly-L-ornithine/fibronectin-coated chamber slide glasses or 12-well plates and fixed in 4% PFA/PBS for 30 min at room temperature. The samples were rinsed with PBS three times and permeabilized with 0.3% Triton X-100/PBS for 5 min at room temperature. After blocking with Blocking One (Nacalai Tesque, 03953–95) for 15 min at room temperature, the slides were incubated at 4 °C overnight with the following antibodies: mouse anti-β-III tubulin (Sigma–Aldrich, T8660-2ML; 1:500), mouse anti-nestin (Merck Millipore, MAB5326; 1:500), anti-GFAP, and rabbit anti-Iba1 (FUJIFILM, 019–19,741; 1:500). After washing three times with PBS, the samples were incubated with secondary antibodies conjugated to Alexa 488 (Thermo Fisher Scientific, A-11034), Alexa 555 (Thermo Fisher Scientific, A-21424; 1:500), Alexa 555 (Thermo Fisher Scientific, A-21434; 1:500) or Alexa 555 (Thermo Fisher Scientific, A-21428; 1:500) for 60 min at room temperature and then subjected to nuclear counterstaining with Hoechst 33258 (Sigma–Aldrich, B2883; 10 μg/mL). The samples were analyzed with a FLUOVIEW FV3000 (OLYMPUS) and LSM700 (Carl Zeiss).
At least 100 cells were observed, and this was done for n = 3. All microglia were infected with pseudotyped lentivirus, but no other neurons, astrocytes, or NS/PCs were infected at all, so no quantitative graphs were made.

Kase Y., Sonn I., Goto M., Murakami R., Sato T, & Okano H. (2023). The original strain of SARS-CoV-2, the Delta variant, and the Omicron variant infect microglia efficiently, in contrast to their inability to infect neurons: Analysis using 2D and 3D cultures. Experimental Neurology, 363, 114379.

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Antibody Immunofluorescence Staining Protocol

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The primary antibodies used in this study are listed in Table 1.
The following secondary antibodies were used: Goat anti‐Rabbit IgG, Alexa Fluor Plus 555 (A32732, 1:500, Invitrogen), Goat anti‐Mouse IgG, Alexa Fluor Plus 488 (A32723, 1:500, Invitrogen), Goat anti‐Mouse IgG1, Alexa 488 (A21121, 1:500, Invitrogen), Goat anti‐Mouse IgG2a, Alexa 555 (A21137, 1:500, Invitrogen), Goat anti‐Mouse IgG2b, Alexa 555 (A21147, 1:500, Invitrogen), Goat anti‐Mouse IgG2b, Alexa 647 (A21242, 1:500, Invitrogen), Goat anti‐Chicken IgY, Alexa Fluor Plus 488 (A32931, 1:500, Invitrogen), Goat anti‐Chicken IgY, Alexa Fluor 647 (A21449, 1:500, Invitrogen), Goat anti‐Rat IgG, Alexa Fluor 647 (ab 150159, 1:500, Abcam), Donkey anti‐Rabbit IgG, Alexa Fluor Plus 647 (A32795, 1:500, Invitrogen), Donkey anti‐Mouse IgG, Alexa Fluor Plus 488 (A32766, 1:500, Invitrogen), Donkey anti‐Goat IgG, Alexa Fluor Plus 488 (A32814, 1:500, Invitrogen), Donkey anti‐Goat IgG, Alexa Fluor Plus 555 (A32816, 1:500, Invitrogen), Donkey anti‐Chicken IgY, Alexa Fluor 647 (703‐605‐155, 1:500, Jackson Immuno‐Research).

Hosoya M., Fujioka M., Murayama A.Y., Ozawa H., Okano H, & Ogawa K. (2021). Neuronal development in the cochlea of a nonhuman primate model, the common marmoset. Developmental Neurobiology, 81(8), 905-938.

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Fluorescent Labeling of CLC-ec1 Protein

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Upon arrival at the Jayaraman lab, the CLC-ec1 protein was diluted 1:10 in a buffer consisting of 10 mM HEPES pH 7.5, 150 mM NaCl, 5 mM Decyl Maltoside, and 1 mM EDTA. Stock solutions of 1 mM maleimide-linked donor and acceptor fluorophores, Alexa 555 and Alexa 647 (Invitrogen), respectively, in DMSO were pre-mixed in a separate tube and then added to the diluted protein to achieve a concentration of 600 nM Alexa 555 and 2.4 µM Alexa 647 (final DMSO <2% in the labeling reaction). The sample was then rotated at room temperature for 30 minutes while being protected from ambient light to allow the fluorophores to label the CLC protein. Labeled protein was again diluted 1:4 in a buffer consisting of 10 mM HEPES pH 7.5, 150 mM NaCl, 5 mM Decyl Maltoside, and 1 mM EDTA, and the resulting labeled CLC protein was used to prepare slides for smFRET.

Cheng R.C., Krishnamoorti A., Berka V., Durham R.J., Jayaraman V., & Maduke M. (2020). Single-molecule FRET monitors CLC transporter conformation and subunit independence.

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Visualizing Cell Migration and Cytoskeletal Dynamics

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Cells cultured on FN-coated glass covers were fixed with 4% paraformaldehyde, permeabilized with 0.1% TritonX-100 and incubated with primary antibodies, followed by Alexa 488-conjugated secondary antibody (Molecular Probes, Eugene, OR). Actin cytoskeleton was visualized using either Alexa 555- (Molecular Probes) or Texas red (Sigma)-conjugated phalloidin. Fluorescence images were obtained using an Olympus confocal microscope (FV1000) with FV10-MSASW software. To assess migration of individual cells, 1×10⁴ cells adapted to microscopic recording media (phenol red-free DMEM containing 5 μM mitomycin C and 15 mM HEPES) were plated on a cover glass in an incubation chamber. Time-lapse movies were recorded over a period of 9 h (Figure 4E and 4F, and supplementary movie SM1) and each frame acquired every 10 min using a microMax cooled EMCCD camera mounted on a Olympus IX81 microscope (Olympus, Tokyo, Japan). Migration paths were traced from nuclear positions, and velocity calculated using Metamorph software (Ver. 6.3r6, Molecular Devices LLC, Sunnyvale, CA). Cellular translocation of GFP-Swiprosin-1 to the lamellipodial leading edge was observed by recording for 10 min every 5 sec (Figure 5A and supplementary movie SM2) using confocal microscopy.

Huh Y.H., Oh S., Yeo Y.R., Chae I.H., Kim S.H., Lee J.S., Yun S.J., Choi K.Y., Ryu J.H., Jun C.D, & Song W.K. (2015). Swiprosin-1 stimulates cancer invasion and metastasis by increasing the Rho family of GTPase signaling. Oncotarget, 6(15), 13060-13071.

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Drosophila Ovary Immunohistochemistry

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The following primary antibodies were used: guinea-pig anti-Tj (G5 or GP6, 1:5000) [57 (link)], rat anti-Bab2 (R10, 1:3000; or R7, 1:2000) [20 (link)], rabbit anti-Vasa (1:2000) [80 (link)], rabbit anti-Vasa (d-260, 1:500; Santa Cruz Biotechnology), chicken anti-Vasa (1:5000; gift from K. Howard and M. Van Doren), rabbit anti-α-spectrin (#254, 1:1000; gift from D. Branton), mouse anti-LamC (LC28.26, 1:50), mouse anti-Hts (1B1, 1:5), mouse anti-N (C17.9C6, 1:5; C458.2H, 1:5), mouse anti-Dl (C594.9B, 1:5), mouse anti-Engrailed (4D9, 1:5), and mouse anti-ßPS integrin (CF.6G11, 1:10) (Developmental Studies Hybridoma Bank), rabbit anti-pMad (PS1, 1:250; gift from T. Tabata) [81 (link)], rabbit anti-pMad (pSmad1/5, 41D10, 1:100; Cell Signalling), rabbit anti-ß-galactosidase (1:1500; MP Biomedicals), and rabbit anti-GFP (1:100; BD Biosciences). Secondary antibodies (1:400) were conjugated either to Cy3, Cy5 (Jackson Immuno Research Laboratories), Alexa-405, Alexa-555, Alexa-488, or Alexa-647 (Molecular Probes, Life Technologies). Ovaries were mounted in Vectashield (Vector Laboratories).

Panchal T., Chen X., Alchits E., Oh Y., Poon J., Kouptsova J., Laski F.A, & Godt D. (2017). Specification and spatial arrangement of cells in the germline stem cell niche of the Drosophila ovary depend on the Maf transcription factor Traffic jam. PLoS Genetics, 13(5), e1006790.

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Cell Adhesion and Cytoskeletal Dynamics

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Fibronectin-coated coverslips were seeded with cryo, live, or Cytochalasin D-treated MSCs at similar concentrations. At 2 hr postincubation, unbound cells were washed away. The cells were fixed with 10% formalin (Fisher Scientific) for 10 min. The fixed cells were washed three times with PBS. F-actin and nuclei were stained with phalloidin conjugated with Alexa 555 (Molecular Probes, Invitrogen) and DAPI (Molecular Probes, Invitrogen). The stained cells were imaged with a Zeiss LSM 510 microscope. Images were analyzed with the use of Zeiss ZEN 2009 software.

Chinnadurai R., Garcia M.A., Sakurai Y., Lam W.A., Kirk A.D., Galipeau J, & Copland I.B. (2014). Actin Cytoskeletal Disruption following Cryopreservation Alters the Biodistribution of Human Mesenchymal Stromal Cells In Vivo. Stem Cell Reports, 3(1), 60-72.

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Standard Western Blotting and Immunofluorescence

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Standard Western blotting analyses were carried out, as described^{7 (link),15 (link),16 (link),25 (link)}. In brief, protein sample preparations were quantified (BCA protein assay; ThermoFisher Scientific) and 40 μg of each sample was loaded onto SDS-polyacrylamide gels, followed by electrophoresis under reducing conditions. Following electroblotting separated proteins onto nitrocellulose membranes (BioRad), the membranes were probed with a specific mouse or rabbit antibody, followed by washing and probed with a secondary antibody conjugated with horseradish peroxidase (ThermoFisher Scientific). Where indicated, immunofluorescence microscopy using specific primary mouse or rabbit antibodies was carried out^{7 (link),15 (link),16 (link),25 (link)}. Secondary antibody against the primary mouse or rabbit antibodies was conjugated with red fluorescent rhodamine, Texas red, or Alexa 555 (Molecular Probes/Invitrogen or Jackson Laboratory). Alternatively, the green fluorescent secondary antibody was conjugated with fluorescein or Alexa Fluor 488 (Molecular Probes/Invitrogen or Jackson Laboratory). Appropriate secondary antibody combinations were matched for dual antibody staining to avoid cross-react^{15 (link)–17 (link)}.

Chen Y.A., Sie Y.D., Liu T.Y., Kuo H.L., Chou P.Y., Chen Y.J., Lee K.T., Chen P.J., Chen S.T, & Chang N.S. (2021). Normal cells repel WWOX-negative or -dysfunctional cancer cells via WWOX cell surface epitope 286-299. Communications Biology, 4, 753.

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Quantitative Immunohistochemistry of PAI-1 in Carotid Arteries

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The carotid segments were perfusion fixed in situ via cardiac puncture at 80 mmHg with an initial infusion of saline with 14.8 mM KCl, followed by 4% phosphate-buffered formalin. After 24–48 h, the artery segments were dehydrated in graded ethanol baths, immersed in citrisolv, and embedded in paraplast (Oxford, St Louis, MO, USA). Transverse sections (3 μm-thick) of the tissue were affixed onto slides positively charged. The slides were submitted to deparaffinization in xylenes and then rehydrated. Heat-induced antigen retrieval was done with Tris-EDTA buffer (pH 9.0) using pressure cooker. Slides were incubated in blocking buffer (Tris Buffer and 1% casein) for 1h. PAI-1 (Santa Cruz) antibody was used at 1:50 dilution overnight at 4°C. After wash steps with Tris-buffer, slides were incubated with ALEXA 555 (Molecular Probes) for 2h and nucleus stained with diamino-2-phenylindole (DAPI) at 10μg/μL. Immunohistochemistry were analyzed on Laser Scanning Microscope 510 (Carl Zeiss). Images were quantified by IMAGE J software.

Miyakawa A.A., Girao-Silva T., Krieger J.E, & Edelman E.R. (2018). RAPAMYCIN ACTIVATES TGF RECEPTOR INDEPENDENTLY OF ITS LIGAND: IMPLICATIONS FOR ENDOTHELIAL DYSFUNCTION.. Clinical science (London, England : 1979), 132(4), 437-447.

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Western Blotting and Immunofluorescence Antibody Protocols

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All the primary antibodies used in this study are detailed in Supplementary Table S2, including vendors' information and the application and dilution conditions. Manufacturers' validation criteria were used for the application of all antibodies. For western blotting, HRP-conjugated goat anti-rabbit and anti-mouse secondary antibodies were purchased from BioRad and used at 1:1000 dilution in 5% BSA or milk. For indirect immunofluorescence staining, fluorescent-conjugated secondary antibodies Alexa-488, Alexa-555 and Alexa-633 were purchased from Molecular Probes (Invitrogen) and used at 1:1000 dilution.

Abdisalaam S., Mukherjee S., Bhattacharya S., Kumari S., Sinha D., Ortega J., Li G.M., Sadek H.A., Krishnan S, & Asaithamby A. (2022). NBS1-CtIP–mediated DNA end resection suppresses cGAS binding to micronuclei. Nucleic Acids Research, 50(5), 2681-2699.

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