Alexa fluor 555 anti rabbit

Manufactured by Thermo Fisher Scientific

Sourced in United States

Alexa Fluor 555 anti-rabbit is a fluorescent secondary antibody that specifically binds to rabbit primary antibodies. It is designed for use in various immunoassay techniques, such as immunohistochemistry, flow cytometry, and Western blotting, to detect and visualize the presence of target proteins or antigens.

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37 protocols using alexa fluor 555 anti rabbit

Immunostaining of Cellular Markers

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The following antibodies were applied: Tetraspanin-4 (NBP1-59438, Novus); RPE 65 (MA1-16578; Thermo Fisher Scientific); GFAP (ab279290, Abcam); CD11b (ab8878, Abcam); integrin α5 (ab288767, Abcam); EOGT (ab190693, Abcam); NDST1 (26203-1-AP, Proteintech); Alix (2171, Cell Signaling); Alix (92,880, Cell Signaling); Smad2/3 (3102, Cell Signaling); Phospho-Smad2/Smad3 (8828, Cell Signaling); Alpha-Smooth Muscle Actin (MA5-11547, Thermo Fisher Scientific); GAPDH (5174, Cell Signaling) and β-actin Rabbit antibodies (ab8227, Abcam). Secondary antibodies used included the following: Alexa Fluor 488 Anti-Mouse; Alexa Fluor 555 Anti-Rabbit; Alexa Fluor 555 Anti-Mouse; and Alexa Fluor 555 Anti-Rabbit (Thermo Fisher Scientific). Additionally, CCK8 (ab228551, Abcam), Dulbecco’s modified Eagle’s medium (DMEM)/F12 culture media, and fetal bovine serum were obtained from Thermo Fisher Scientific.

Wu L., Yang S., Li H., Zhang Y., Feng L., Zhang C., Wei J., Gu X., Xu G., Wang Z, & Wang F. (2022). TSPAN4-positive migrasome derived from retinal pigmented epithelium cells contributes to the development of proliferative vitreoretinopathy. Journal of Nanobiotechnology, 20, 519.

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Whole-cell and LFP Recordings in Hippocampus

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For LFP recordings, glass microelectrodes (tip diameter ~5–10 μm; resistance: 0.2–0.3 MΩ) were filled with ACSF before use. Whole-cell recordings were performed with borosilicate glass electrodes (2–5 MΩ) filled with (in mM) 120 K-gluconate, 10 HEPES, 10 KCl, 3 Mg-ATP, 5 EGTA, 2 MgSO₄, 0.3 Na-GTP and 14 phosphocreatine. The pH was adjusted to 7.4 with KOH. LFP signals in the CA3 pyramidal cell layer were amplified 1,000-fold, filtered (1–8 kHz), and sampled at 20 kHz. Whole-cell and extracellular recordings were performed using a Multiclamp 700A amplifier (Axon Instruments). For parallel double patch-clamp and field recordings, a custom-made two channel extracellular amplifier was used. Cells were routinely loaded with 0.2% biocytin. After recordings, slices were transferred to 4% paraformaldehyde. Biocytin-filled cells were subsequently visualized with streptavidin conjugated with Dy-Light 488. After acquisition of confocal images, neuronal reconstruction was performed with the imageJ package (Schneider et al., 2012 (link)). To better estimate the slice position along the dorso-ventral axis slices were either Nissl stained or stained with anti-NeuN (Millipore) or anti-calbindin (Swant) antibodies followed by the secondary polyclonal antibody anti-mouse Alexa Fluor 488 or anti-rabbit Alexa Fluor 555 (TermoFisher), respectively.

Imbrosci B., Nitzan N., McKenzie S., Donoso J.R., Swaminathan A., Böhm C., Maier N, & Schmitz D. (2021). Subiculum as a generator of sharp wave-ripples in the rodent hippocampus. Cell reports, 35(3), 109021.

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Whole-cell and LFP Recordings in Hippocampus

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Quantifying Amisyn and Dynamin1 Expression

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To quantify the overexpression, INS-1 cell were transfected with either Cherry2-amisyn or Dynamin1-GFP, fixed 24 hr later in 3.8% formaldehyde in phosphate-buffered saline (PBS) for 30 min at 25°C and washed in PBS. The cells were permeabilized in 0.2% Triton X-100 in PBS for 5 min and washed in PBS. Blocking was done using 5% FBS in PBS for 1–2 hr at 25°C. Cells were then incubated with a primary antibody (anti-Dynamin1, ab52852 abcam or anti-Amisyn, ab153974 abcam) both diluted 1/50 in 5% FCS in PBS over night at 4°C and washed again in PBS. Incubation with secondary antibody (Alexa Fluor 488 anti-rabbit or Alexa Fluor 555 anti-rabbit, Invitrogen) diluted 1/1000 in 5% FCS in PBS was performed for 1 hr at 25°C and subsequently the cells were washed in PBS.

Guček A., Gandasi N.R., Omar-Hmeadi M., Bakke M., Døskeland S.O., Tengholm A, & Barg S. (2019). Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis. eLife, 8, e41711.

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Immunohistochemical Staining of Neural Markers

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50 um fixed slices were stained using the following primary antibodies: rabbit polyclonal TH (1:500; Chemicon) or rabbit polyclonal Vgat (1:1000; Synaptic Systems) and goat polyclonal GFP (1:300; Abcam). Secondaries were Alexa Fluor 488 anti-goat (Molecular Probes) and Alexa Fluor 555 anti-rabbit (Invitrogen). Slices were DAPI-stained and mounted on slides, and images were captured using a Nikon NIS microscope or a Zeiss Axioscan slide scanner.

Harris J.J., Kollo M., Erskine A., Schaefer A, & Burdakov D. (2022). Natural VTA activity during NREM sleep influences future exploratory behavior. iScience, 25(6), 104396.

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Immunofluorescence Imaging of Cytoskeletal Proteins

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HEK293 cells were grown on round glass coverslips (Fisher Scientific) in 35 mm cell culture dishes. After treatment with 1, 2, 4, or 19 for 4 h, the cells were fixed with pre-chilled methanol for 20 min. The coverslips were washed with phosphate-buffered saline (PBS), and blocked with 2% bovine serum albumin (BSA) in PBS for 30 min and then incubated with primary antibody (Texas Red-antiphalloidin, Life Tecnologies; antivimentin, Sigma; antiannexin II – Santa Cruz) for 1 h (see Western Blot Analyses section for discussion of antibodies). After removal, the coverslips were washed three times for 5 min washes with PBS and then incubated with a fluorescently labeled secondary antibody (Alexa Fluor 647 antimouse and Alexa Fluor 555 antirabbit, Invitrogen) for another 50 min. After washing with PBS, the coverslips were mounted onto glass slides with anti-fade mounting medium (Invitrogen). Images were captured with a Zeiss Observer Z1 microscope by using Slidebook 4.2.0.11 (Intelligent Imaging Innovations).

Tao S., Tillotson J., Wijeratne E.M., Xu Y.M., Kang M., Wu T., Lau E.C., Mesa C., Mason D.J., Brown R.V., Clair J.J., Gunatilaka A.A., Zhang D.D, & Chapman E. (2015). Withaferin A Analogs That Target the AAA+ Chaperone p97. ACS chemical biology, 10(8), 1916-1924.

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DRG Cryosectioning and Immunohistochemistry

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L4-L5 DRG tissue were extracted, fixed in 4% paraformaldehyde dissolved in PBS, cryoprotected in 30% sucrose, and frozen in OCT (Tissue-Tek). Ten-μm thick cryosections were blocked with 1% bovine serum albumin (Sigma-Aldrich)/0.1%Triton X-100 in 0.1 M phosphate buffered saline (PBS) and then incubated with primary antibodies overnight at 4°C. After 3 washes in PBS for 10 minutes each, sections were incubated with secondary antibodies for 1 hour at room temperature, washed 3 times in PBS (10 minutes each) and mounted using Dako mounting medium (S3023). The following primary antibodies were used: Isolectin B4 (IB4) DyLight 594-conjugated (Vector laboratories FL-1207); CGRP (Millipore; PC205L), DDX3X (santa cruz; sc-365768); NF200 (Abcam; ab4680); Parvalbumin (Swant; PV-25). Secondary antibodies used: Alexa Fluor 555 anti-rabbit (Invitrogen; A32794), Alexa Fluor 555 anti-mouse (Invitrogen; A32773); Alex Fluor 488 anti-rabbit (Invitrogen; A21206); beta-tublin-488 (BD Pharmingen; 560338).

Cronin S.J., Tejada M.A., Song R., Laval K., Cikes D., Ji M., Brai A., Stadlmann J., Novatchikova M., Perlot T., Ali O.H., Botta L., Decker T., Lazovic J., Hagelkruys A., Enquist L., Rao S., Koyuncu O.O, & Penninger J.M. (2023). Pseudorabies virus hijacks DDX3X, initiating an addictive “mad itch” and immune suppression, to facilitate viral spread. bioRxiv.

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Immunostaining of Mitochondrial Proteins in HeLa Cells

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HeLa cells were cultured on glass slides (12 mm, VWR), fixed with 3.7% Formaldehyde (AppliChem Panreac ITW companies), permeabilised with 0.5% Triton X-100 (Sigma-Aldrich), washed shortly with 0.05% Tween-20 (Merck) and blocked with 5% goat serum (Sigma-Aldrich) to avoid unspecific binding. For immunostaining slides were incubated for 1 h with the following primary antibodies (all from Santa Cruz): rabbit-anti-human mtSSB (sc-67101 [98 (link)]), TWINKLE (sc-134915 [99 (link)]) and APE1 (sc-334 [54 (link)]) as well as mouse-anti-human MFN2 (sc-100560 [100 (link)]) and OPA1 (sc-393296 [101 (link)]). Alexa Fluor 555 anti-mouse, Alexa Fluor 555 anti-rabbit and Alexa Fluor 488 anti-rabbit (all from Invitrogen) were used as secondary antibodies. HeLa cells were mounted with Vectashield^® containing 4′,6-Diamidin-2-phenylindol (DAPI, Vector laboratories) to stain the nuclei and imaged using a Keyence BZ-9000 microscope (Keyence Germany). The fluorescence intensity was analysed using the ImageJ 1.46/1.51 software (National Institutes of Health).

Wiehe R.S., Gole B., Chatre L., Walther P., Calzia E., Ricchetti M, & Wiesmüller L. (2018). Endonuclease G promotes mitochondrial genome cleavage and replication. Oncotarget, 9(26), 18309-18326.

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Immunofluorescence Staining Protocol

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Alexa Fluor 647-coupled antibody against γH2A.X protein was obtained from Becton Dickinson (Heidelberg, Germany). Anti-53BP1 rabbit polyclonal antibody was purchased from Santa Cruz (Heidelberg, Germany). Secondary antibody Alexa Fluor 555 (anti-rabbit) and Hoechst33342 were purchased from Invitrogen (Eugene, OR, USA). DAKO Fluorescent mounting medium from Dako North America Inc. (Carpinteria, CA, USA) was used. All media, fetal bovine serum (FBS) and penicillin-streptomycin (pen/strep) were acquired from Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals were purchased from Sigma-Aldrich (Deisenhofen, Germany) unless otherwise specified.

Szymonowicz K., Krysztofiak A., van der Linden J., Kern A., Deycmar S., Oeck S., Squire A., Koska B., Hlouschek J., Vüllings M., Neander C., Siveke J.T., Matschke J., Pruschy M., Timmermann B, & Jendrossek V. (2020). Proton Irradiation Increases the Necessity for Homologous Recombination Repair Along with the Indispensability of Non-Homologous End Joining. Cells, 9(4), 889.

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Immunostaining of Drosophila Neural Tissues

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Brains and VNC of 5–7-days-old flies were dissected in dissection buffer (PBST: 0.015% triton X-100 in 1x PBS) and fixed in 4% PFA at room temperature on a shaker for 20 min, then washed for 4 × 20 min in wash buffer (0.3% triton in 1x PBS). After this, the tissues were blocked in block buffer (1x heat inactivated normal goat serum with 0.3% triton in 1x PBS) for 30 min at room temperature. The samples were then incubated with primary antibody at 4 °C overnight. The primary antibodies used were: Rabbit-GFP (Invitrogen, A11122, 1:500 dilution), Rabbit-DsRed (Rockland 39707. 1:500 dilution), Mouse-GFP (Sigma-Aldrich, G6539. 1:500 dilution), Chicken-GFP (AVES, GFP-1020. 1:500 dilution), Rabbit-GABA (Sigma-Aldrich, A2052, 1:500 dilution), Mouse-nc82 (Hybridoma Bank DSHB, Brunchpilot, 1:50 dilution). On the second day, tissues were washed for 4 × 20 min and then incubated in secondary antibodies. The secondary antibodies were all from Invitrogen and used at 1:200 dilution: Alexa Fluor 555 anti-Rabbit (A-21428), Alexa Fluor 488 anti-Mouse (A11001), Alexa Fluor 647 anti-Mouse (A-2123511031). After incubation, tissues were washed for 4 × 10 min. VNC and brains were mounted on a slide for imaging. An Olympus FV1000 microscope with 20X air lens or 40X oil-immersion lens was used for confocal imaging.

Liu C., Zhang B., Zhang L., Yang T., Zhang Z., Gao Z, & Zhang W. (2020). A neural circuit encoding mating states tunes defensive behavior in Drosophila. Nature Communications, 11, 3962.

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