Alexa fluor 555 secondary antibody

Manufactured by Thermo Fisher Scientific

Sourced in United States, China

Alexa Fluor 555 secondary antibody is a fluorescent-labeled secondary antibody used in immunoassays. It is designed to bind to primary antibodies and emit light at a specific wavelength, enabling the detection and visualization of target proteins or molecules.

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Lab products found in correlation

Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (11 mentions) Alexa fluor 488, by Thermo Fisher Scientific (8 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions) Metaxpress neurite outgrowth application module, by Molecular Devices (4 mentions) Lsm 710, by Zeiss (4 mentions) Lsm 710 confocal microscope, by Zeiss (3 mentions) Slowfade antifade, by Thermo Fisher Scientific (3 mentions) Lsm 5 3.2 image capture and analysis software, by Zeiss (3 mentions) Lsm 510 confocal microscope, by Zeiss (3 mentions) Sytox green, by Thermo Fisher Scientific (3 mentions) Anti nf 160 primary antibody, by Merck Group (3 mentions) Nanozoomer ht, by Hamamatsu Photonics (3 mentions) Lsm 700 confocal microscope, by Zeiss (2 mentions) Axiovision software, by Zeiss (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions) Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions) Np 40, by Merck Group (2 mentions) Lsm 800, by Zeiss (2 mentions) Lsm 880, by Zeiss (2 mentions)

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68 protocols using alexa fluor 555 secondary antibody

Immunohistochemical Analysis of Neuronal Markers

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Mice were perfused using 0.9% saline and then 4% paraformaldehyde. The mouse brains were fixed in 4% paraformaldehyde for 48 h and then in 30% sucrose for 48 h. A microtome was used to cut 30-μm-thick coronal sections. The sections were permeabilized in blocking solution (Tris-buffered saline containing 10% normal goat serum (Invitrogen), 2% BSA and 0.4% Triton X-100, both from Fisher Scientific) at room temperature for 2 h. Next, the sections were incubated in blocking solution containing NeuN antibody (1:500) overnight at 4°C. Incubation with primary antibody was followed by incubation with blocking solution containing Alexa Fluor 555 secondary antibody (Life Technologies, 1:500) for 2 h at room temperature. Before mounting the brain sections onto glass, sections were incubated in PBS containing Hoechst stain (1:10,000, Invitrogen) for 10 min.
Overview images were acquired using a Nikon Eclipse E800 microscope using ×4/0.2 NA Plan Apo objective with an Optimus sCMOS camera. 4′,6-diamidino-2-phenylindole/TRITC channels were used to take images. Images of the CA1 region were acquired using a Leica SP8 scanning confocal microscope using ×40/1.30 NA HC PI Apo CS2 oil immersion objective with a PCO Edge 5.5 sCMOS camera. Wave solid-state lasers at 405 and 561 nm were used for illumination. The images were analyzed using Fiji software (34 (link)).

Pitera A.P., Asuni A.A., O'Connor V, & Deinhardt K. (2019). Pathogenic tau does not drive activation of the unfolded protein response. The Journal of Biological Chemistry, 294(25), 9679-9688.

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Lung Inflammation and CCR4 Expression Analysis

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To assess lung inflammation, the left lung was infused via the mainstem bronchus with neutral buffered formalin at 4 h after resuscitation and harvested. Following fixation, tissue specimens were embedded in paraffin, sectioned and stained. After hematoxylin and eosin staining, lungs were evaluated with light microscopy. Inflammatory cells were identified using a specific monoclonal rat anti-mouse Ly-6B.2 alloantigen antibody (1:500, AbD Serotec, Raleigh NC) following antigen retrieval. For detection, horseradish peroxidase (HRP; BD Pharmingen, San Diego, CA) and Ultravision Detection System-DAB Plus Substrate System (Fremont, CA) were used. Inflammatory cells were quantified by blindly counting positively stained cells within ten high-powered fields per mouse.
In order to determine CCR4 expression in pulmonary tissue, sections were stained with a polyclonal antibody against mouse CCR4 (1:1000, AbCam, Cambridge, MA) and DAB staining. CCR4 expression was identified on cultured human bronchial epithelial cells with an anti-human CCR4 monoclonal antibody (1:100, R&D Biosystems) and an AlexaFluor^®-555 secondary antibody (1:1000, Life Technologies, Grand Island, NY). For CCR4 immunostaining, IgG controls were used to confirm antibody specificity.

Richter J.R., Sutton J.M., Belizaire R.M., Friend L.A., Schuster R.M., Johannigman T.A., Miller S.G., Lentsch A.B, & Pritts T.A. (2014). Macrophage-Derived Chemokine (MDC/CCL22) is a Novel Mediator of Lung Inflammation Following Hemorrhage and Resuscitation. Shock (Augusta, Ga.), 42(6), 525-531.

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Characterization of Cancer Stem Cells

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Cells were grown in 6‐well plates (10⁵ cells per well). After 48 h, cells were digested, counted, and stained with antibodies: anti‐CD44‐FITC (BD, 1:100) and anti‐CD24‐PE (BD, 1:100). ALDH enzymatic activity was assessed using an ALDEFLUOR kit (Promega) according to the provided manual. For the detection of EGFR on the membrane, when the cells had grown to 80% confluence, NH₄Cl was added into the medium for 4 h and the cells were trypsinized. Cells were then blocked with 5% goat serum for 10 min. Cells were first stained with EGFR (Abcam, ab30) for 60 min on ice and then washed with phosphate‐buffered saline (PBS) followed by incubation with Alexa Fluor 555 secondary antibody (Life Technologies, 1:1000) on ice for 30 min.

Zhao L., Qiu T., Jiang D., Xu H., Zou L., Yang Q., Chen C, & Jiao B. (2020). SGCE Promotes Breast Cancer Stem Cells by Stabilizing EGFR. Advanced Science, 7(14), 1903700.

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Adenosine Receptor Inhibitors and Pluripotency

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In order to investigate the role of adenosine receptors in pluripotency, we cultivated and treated ESC with the inhibitors of ALPL (1 mM levamisole), CD73 (α,β-methyleneadenosine 5′-diphosphate sodium salt), and antagonists of the A1R (10 nM PSB 36), A2AR (645 nM ANR 94), A2BR (10 nM PSB 603), A3R (50 nM MRS 3777), and the less specific inhibitor, caffeine (50 μM). After 96 h, the cells were washed, trypsinized, and fixed with a 4% paraformaldehyde solution. The cells were washed in phosphate-buffered saline (PBS) and blocked with a 4% normal donkey serum and 0.1% Triton-X in PBS for 60 min. The cells were then incubated with mouse anti-SSEA1 antibody (Millipore, 1:500 dilution) for 2 h. In sequence, the cells were washed with a 4% normal donkey serum and 0.1% triton-x in PBS and incubated with Alexa Fluor 555 secondary antibody (Life Technologies, 1:500) for 1 h. As the negative control, we used the cells that had undergone the whole process but were not labeled by the primary antibodies. The percentage of marked cells was measured in the Attune^® flow cytometer (Thermo Fisher Scientific). At least 50,000 events were acquired per sample, and the analysis was performed in FlowJo software.

Glaser T., Martins P., Beco R., Bento C.A., Cappellari A.R., La Banca Oliveira S., Merkel C.A., Arnaud-Sampaio V.F., Lameu C., Battastini A.M, & Ulrich H. (2024). Impairment of adenosine signaling disrupts early embryo development: unveiling the underlying mechanisms. Frontiers in Pharmacology, 14, 1328398.

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Apoptosis Analysis in H9c2 Cells

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Immunostaining was performed as described in detail in our previous studies (Tseng A et al., 2010 (link); Zhang L et al 2012 (link)). H9c2 cardiomyoblasts were fixed via 4% paraformaldehyde and then blocked with the incubation of polyclonal anti-active caspase 3 antibody (Abcam, Cambridge, MA), which was followed by goat anti-rabbit Alexa Fluor 555 secondary antibody (Life Technologies). The percentage of apoptotic positive cells was determined in five randomly chosen fields and was normalized with the total number of stained nuclei by DAPI.

Wang H., Zhao Y.T., Zhang S., Dubielecka P.M., Du J., Yano N., Chin Y.E., Zhuang S., Qin G, & Zhao T.C. (2017). Irisin plays a pivotal role to protect the heart against ischemia and reperfusion injury: A novel approach to inducing cardioprotection. Journal of cellular physiology, 232(12), 3775-3785.

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Immunofluorescence analysis of myotube diameter

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Cells were washed for 2 × 5 min in PBS and fixed in 4% paraformaldehyde for 15 min. Cells were then washed in PBS (3 × 5 min), permeabilized with 0.1% Triton X-100 for 10 min, washed in PBS (3 × 5 min) and blocked in 3% (w/v) BSA in PBS for 1 h at RT. Cells were incubated overnight at 4ºC in anti-sarcomeric myosin (1:50 diluted in 3% BSA/PBS, MF 20, developed by D.A. Fischman, Weill Cornell Medical College, obtained from the Developmental Studies Hybridoma Bank, Iowa City, IA, USA). The following day, cells were washed in PBS (4 × 5 min) and incubated in goat-anti-mouse Alexa Fluor 555 secondary antibody (1:400, Life Technologies) and DAPI (1:1,000) for 2 h at RT. Cells were washed in PBS (4 × 5 min) and then imaged on a Zeiss Axiovert 40 CFL inverted microscope using a 20× objective to give a total magnification of 126×. Five images were taken in each well from pre-defined locations within each quadrant. Myotube diameter was quantified using AxioVision AC software (AxioVision AC release 4.8.2, Carl Zeiss, Wrek, Göttingen, Germany).

Murphy K.T., Hossain M.I., Swiderski K., Chee A., Naim T., Trieu J., Haynes V., Read S.J., Stapleton D.I., Judge S.M., Trevino J.G., Judge A.R, & Lynch G.S. (2018). Mas receptor activation slows tumor growth and attenuates muscle wasting in cancer. Cancer research, 79(4), 706-719.

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Immunostaining for Neuronal Regeneration

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At 3 or 12 weeks after ONC, tissues were collected after perfusion for immunostaining. Frozen sections (25 μm thickness) were prepared using a cryostat microtome (Leica, Deer Park, IL, USA ) and fixed in 4% PBS-buffered paraformaldhyde solution, and permeabilized using 0.3% Triton-X 100 and 3% goat serum in PBS for immunostaining. Tissue samples were incubated with primary antibodies diluted in PBS that contained 3% goat serum at 4 °C overnight. The primary antibodies used were rabbit anti-GAP43 (1:500, Cell Signaling, Denve, MA, USA), anti-β-III tubulin (1:500, Cell signaling) and anti-MBP (1:500, Cell signaling). After washing 3 times with PBS, the samples were incubated with Alexa Fluor 488 (1:200, green) or Alexa Fluor 555 secondary antibody (1:200, red) (Molecular Probes, Waltham, MA, USA) for 1 h at room temperature. The samples were mounted in fluoro mounting medium (Millipore, Burlington, MA, USA) and images were taken with a Zeiss LSM700 confocal microscope (Kuehnstrasse, Hamburg, Germany). The fluorescence images were analyzed using ImageJ from the National Institute of Health (Bethesda, MD, USA) to analyze the fluorescence intensities of the tissues compared with that of the control after subtracting the background value.

Kwon H.S., Kevala K., Qian H., Abu-Asab M., Patnaik S., Marugan J, & Kim H.Y. (2023). Ligand-Induced Activation of GPR110 (ADGRF1) to Improve Visual Function Impaired by Optic Nerve Injury. International Journal of Molecular Sciences, 24(6), 5340.

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Immunodetection of BoNT-A-truncated SNAP-25

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Sections corresponding to the TNC region of the brainstem were randomly selected, washed in PBS with 0.25% Triton X-100 (PBST), and exposed to 50 μL of 3% peroxide diluted in 200-μL PBS for 1h. Sections were washed again and blocked with 10% normal goat serum (NGS) in PBST. A well-characterized rabbit polyclonal antibody that specifically cleaves the BoNT-A-truncated SNAP-25 (nonaffinity-purified rabbit antiserum (anti-SNAP-25₁₉₇, National Institute for Biological Standards and Control, Potters Bar, UK, a generous gift from Dr. Thea Sesardic) was diluted in 1% NGS (1:4000), and samples were incubated overnight at room temperature. The next day, the sections were washed, and 200 μL of Alexa Fluor 555 secondary antibody (1:400, Molecular Probes, Invitrogen, Carlsbad, CA, USA) was used to enhance the cSNAP-25 signal. Samples were then washed 3 times before mounting into the glass slide and coverslipped with an antifading agent. All samples were visualized with an epifluorescent microscope (Olympus BX-51, Tokyo, Japan). Fiber-shaped staining was searched, and pictures were taken using a digital camera (DP-70, Olympus, Tokyo, Japan).

Muñoz-Lora V.R., Dugonjić Okroša A., Matak I., Del Bel Cury A.A., Kalinichev M, & Lacković Z. (2022). Antinociceptive Actions of Botulinum Toxin A1 on Immunogenic Hypersensitivity in Temporomandibular Joint of Rats. Toxins, 14(3), 161.

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Quantifying Adipocyte Size and Browning

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Subcutaneous adipose tissue was removed and was fixed in 10% buffered neutral formalin. It was then embedded in paraffin, sectioned and stained. All adipose sections were viewed at 40X magnification, and images were captured using Zeiss microscope connected via camera to a computer (progres® capture pro 2.1 camera). Adipocytes size measurement was performed using a computer-assisted image analysis progres® capture pro software. From each animal 10 images were measured. The area of each adipocyte was measured by tracing the cell boundary on the images captured. All adipocytes that had complete cell boundary were measured. Data obtained after measurement were averaged to individual animal and later group mean and SEM were calculated.
For PRDM16 and UCP1 immuno staining, subcutaneous adipose sections were de-paraffinized and blocked in 1% BSA in PBS for 30 min at RT and incubated with PRDM16 (ProSci, USA) or UCP1(Abcam, USA) primary antibodies for 90 min at RT, followed by washing with PBS. Sections were then incubated with secondary antibody (goat anti-rabbit Alexa Fluor 555 secondary antibody, Molecular Probes, Invitrogen) for 30 min at RT and mounted using fluromount. Localization of PRDM16 and UCP1 were assessed and the images were captured at 40X magnification.

Anil T.M., Dandu A., Harsha K., Singh J., Shree N., Kumar V.S., Lakshmi M.N., Sunil V., Harish C., Balamurali G.V., Naveen Kumar B.S., Gopala A.S., Pratibha S., Sadasivuni M., Anup M.O., Moolemath Y., Venkataranganna M.V., Jagannath M.R, & Somesh B.P. (2014). A novel 11β-hydroxysteroid dehydrogenase type1 inhibitor CNX-010-49 improves hyperglycemia, lipid profile and reduces body weight in diet induced obese C57B6/J mice with a potential to provide cardio protective benefits. BMC Pharmacology & Toxicology, 15, 43.

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Assessing Neurite Outgrowth in ΔK280 Tau-Expressing SH-SY5Y Cells

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As described, ∆K280 tau_RD-DsRed SH-SY5Y cells were seeded in a 24-well plate (5 × 10⁴/well) with retinoic acid addition on day 1, treated with tested compounds (10 µM) and ∆K280 tau_RD-DsRed expression was induced with doxycycline (2 μg/mL) on day 2. On day 8, after being fixed in 4% paraformaldehyde for 15 min, permeabilized in 0.1% Triton X-100 for 10 min and blocked in 3% bovine serum albumin (BSA) for 20 min, the cells were stained with TUBB3 (neuronal class III β-tubulin) primary antibody (1:1000; BioLegend, San Diego, CA, USA) at 4 °C overnight, followed by goat anti-rabbit Alexa Fluor^® 555 secondary antibody (1:1000; Molecular probes) at room temperature for 2 h, with 4′-6-diamidino-2-phenylindole (DAPI, 0.1 µg/mL; Sigma-Aldrich) included for nuclei staining. Neuronal images were captured using the high-content analysis system as described. Neurite total length (μm), processes (primary neurite extensions projecting directly from the cell body) and branching (points at which primary neurites bifurcated) were analyzed using Neurite Outgrowth Application Module (MetaXpress; Molecular Devices). Around 5000 cells were analyzed in each of three independent experiments for each sample.

Lin T.H., Chang K.H., Chiu Y.J., Weng Z.K., Sun Y.C., Lin W., Lee-Chen G.J, & Chen C.M. (2022). Neuroprotective Action of Coumarin Derivatives through Activation of TRKB-CREB-BDNF Pathway and Reduction of Caspase Activity in Neuronal Cells Expressing Pro-Aggregated Tau Protein. International Journal of Molecular Sciences, 23(21), 12734.

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