Alexa 488 conjugated goat anti mouse antibody

Manufactured by Thermo Fisher Scientific

Sourced in United States

Alexa Fluor 488-conjugated goat anti-mouse antibody is a secondary antibody used for detection in immunological techniques. It is designed to bind to mouse primary antibodies, allowing for visualization and identification of target antigens.

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Alexa 488 (1863 citations) Anti-mouse Alexa-488 (181 citations) Goat anti-mouse Alexa 488 (164 citations) Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (74 citations) Alexa Fluor 488 conjugated goat anti-mouse IgG secondary antibody (56 citations) Alexa 488-conjugated goat anti-mouse (50 citations) Alexa-488-conjugated goat anti-mouse secondary antibody (30 citations) Goat anti-mouse Alexa 488 antibody (25 citations) Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) antibody (13 citations) Alexa Fluor 488 conjugated goat anti-mouse IgG cross-adsorbed secondary antibody (9 citations)

50 protocols using alexa 488 conjugated goat anti mouse antibody

Antibody Staining for TRPV1 in Intestine

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Details of the primary antibodies used in the present study are shown in Table 1. The specificity of anti-TRPV1 antibodies is presented in Supplementary Figures S1 and S2. The secondary antibodies used were FITC-labeled donkey anti-mouse IgG antibody and Cy3-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, United States) for intestinal tissues and Alexa488-conjugated goat anti-mouse antibody and Alexa568-conjugated goat anti-rabbit antibody (Molecular Probes, Eugene, OR, United States) for isolated longitudinal muscle layer-myenteric plexus (LM-MP) and cultured cells.

Yamamoto M., Nishiyama M., Iizuka S., Suzuki S., Suzuki N., Aiso S, & Nakahara J. (2016). Transient receptor potential vanilloid 1-immunoreactive signals in murine enteric glial cells. World Journal of Gastroenterology, 22(44), 9752-9764.

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Visualizing Gln3 Localization in Yeast

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Yeast cells expressing GLN3-myc₁₃ were grown in glutamine or proline medium at 23 or 37°C to early log phase. Cells were shifted to media with different nitrogen conditions. Aliquots of cultures were collected at various time points after the shift and fixed directly in culture medium with 3.7% formaldehyde. After incubation at room temperature for 30 min, cells were transferred to phosphate saline buffer (pH 7.4) containing 3.7% formaldehyde and incubated for additional 30 min. Fixed cells were then prepared for immunofluorescence staining as previously described (Pringle et al., 1991 (link)). Anti-myc (9E10) antibody and Alexa 488-conjugated goat anti‐mouse antibody (Molecular Probes) were used, respectively, as primary and secondary antibodies. Nuclei were stained with DAPI (4′,6‐diamidino‐2‐phenylindole dihydrochloride) prior to mounting. Fluorescent samples were visualized using Olympus FluoView™ FV1000 confocal microscope. For each condition, ~300 cells from three independent experiments were counted and analyzed for Gln3 localization. Cells were randomly selected from at least five different fields for the analysis. Data shown are percentages of cells with nuclear staining of Gln3-myc and expressed as mean ± SD. Comparison analyses between different time-points were done using Student’s t-test. A P-value < 0.05 is considered statistically significant.

Li J., Yan G., Liu S., Jiang T., Zhong M., Yuan W., Chen S., Zheng Y., Jiang Y, & Jiang Y. (2017). Target of rapamycin complex 1 and Tap42-Associated Phosphatases Are Required for Sensing Changes in Nitrogen Conditions in the Yeast Saccharomyces cerevisiae. Molecular microbiology, 106(6), 938-948.

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Analyzing Cofilin Localization in Cells

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Cells were preincubated with 500 nM MitoTracker Red CMXRos (Molecular Probes, Eugene, OR, USA) for 30 min at 37 °C, washed two times with RPMI 1640 medium, and then treated with or without 20 μM AT-101 for 12 h. Cells were then collected via centrifugation, fixed with 4% paraformaldehyde for 15 min, permeabilized by 0.1% Triton X-100 for 10 min, and then blocked with 1% bovine serum albumin for 30 min. Cells were further incubated with a primary cofilin antibody at 4 °C overnight, followed by a secondary Alexa 488-conjugated goat anti-mouse antibody (Molecular Probes) for 1 h at room temperature. After washing, images were captured using a Leica scanning confocal microscope (TCS SP5; Leica Microsystems, Mannheim, Germany).

Li G., Liu L., Shan C., Cheng Q., Budhraja A., Zhou T., Cui H, & Gao N. (2014). RhoA/ROCK/PTEN signaling is involved in AT-101-mediated apoptosis in human leukemia cells in vitro and in vivo. Cell Death & Disease, 5(1), e998-.

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CD31 Immunostaining and EGFR FISH Analysis

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Briefly, slides were blocked with PBS/3% bovine serum albumin (BSA) for 20 minutes and immunostained with mouse anti-human CD31 (1 : 20; Dako Cytomation, France) for 30 minutes at room temperature. Alexa 488-conjugated goat anti-mouse antibody (1 : 1000; Molecular Probes, Invitrogen, France) was added as secondary reagent. After immunostaining, slides were washed three times for 5 minutes each in PBS containing 0.5% Tween-20 (Sigma-Aldrich, France). Only sections of high morphologic quality were used for fluorescent in situ hybridization (FISH). The EGFR FISH Probe Mix (Dako Cytomation, France) was used according to the manufacturer's instructions. Slides were dehydrated through a series of ethanol washes (70%, 90%, and 100%), denatured in the presence of the specific probes at 82°C for 5 minutes, and incubated overnight in a humid chamber at 45°C. Posthybridization washes were performed, and the slides were mounted in antifade medium Fluoromount-G (Interchim, France) with DAPI (Sigma-Aldrich, France). Slides were analyzed using a Zeiss AxioImager.Z1 microscope.

El Hallani S., Colin C., El Houfi Y., Idbaih A., Boisselier B., Marie Y., Ravassard P., Labussière M., Mokhtari K., Thomas J.L., Delattre J.Y., Eichmann A, & Sanson M. (2014). Tumor and Endothelial Cell Hybrids Participate in Glioblastoma Vasculature. BioMed Research International, 2014, 827327.

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VLP Particle Internalization in HeLa Cells

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Three hours after inoculation with 0.256 hemagglutination units of VLP-Z particles or wtVLP particles per cell in 2 ml of DMEM supplemented with 10% fetal bovine serum, HeLa cells in 35-mm glass bottom plates were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature and then 70% methanol for 5 min at 20 ° C. The fixed cells were incubated with a mouse anti-VP 1 antibody (1: 500) [14] and then with Alexa 488-conjugated goat anti-mouse antibody (Molecular Probes, Eugene, Oreg., USA). The cells were examined and scanned using a laser-scanning confocal microscope (Olympus, Tokyo, Japan).

Deng Y.N., Zeng J.Y., Su H, & Qu Q.M. (2015). Recombinant VLP-Z of JC Polyomavirus: A Novel Vector for Targeting Gene Delivery. Intervirology, 58(6).

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Immunofluorescence Staining of Mouse Heart

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For immunofluorescence, the mouse heart tissue paraffin sections were retrieved using pH 9.0 Tris-EDTA. Then, 3% hydrogen peroxide was used to block endogenous peroxidase. The sections were incubated with primary antibodies after blocking sections using CD44 rat antibody (BD, 558739, 1:200), CD31 rabbit antibody (CST, 77699, 1:200), and cTnT mouse antibody (Huabio, EM1701-39, 1:100). Finally, sections were incubated with the secondary antibodies Alexa 647-conjugated goat anti-rabbit antibody (Invitrogen, A32733, 1:500), Alexa 647-conjugated goat anti-rat antibody (Invitrogen, A21247, 1:500), Alexa 488-conjugated goat anti-mouse antibody (Invitrogen, A32723, 1:500), and DAPI.

Zhang Q., Chen L., Huang L., Cheng H., Wang L., Xu L., Hu D., He C., Fu C, & Wei Q. (2022). CD44 promotes angiogenesis in myocardial infarction through regulating plasma exosome uptake and further enhancing FGFR2 signaling transduction. Molecular Medicine, 28, 145.

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CD44 and FGFR2 Colocalization in HUVECs

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HUVECs were plated on fibronectin (10 µg/mL, Thermo Fisher, PHE0023) -coated coverglass in a 48-well cell culture plate. Cells were washed with PBS, fixed with 3% paraformaldehyde, and permeabilized with 0.1% Brij 98. Next, the cells were blocked using 10% goat serum and incubated with CD44 rabbit antibody (Proteintech, 15675-1-AP, 1:200) and FGFR2 mouse antibody (Huabio, M1501-2, 1:50) at 4 °C overnight, followed by Alexa 488-conjugated goat anti-mouse antibody (Invitrogen, A32723, 1:500) and Alexa 647-conjugated goat anti-rabbit antibody (Invitrogen, A32733, 1:500) incubation for 1 h. The cell nuclei were stained with DAPI.
Primary MVECs were cocultured with 10 µg/mL plasma exosomes within 0 min, 15 min, and 30 min. Then, the cells were fixed, permeabilized, and blocked as described above. Next, the cells were incubated with EEA1 mouse antibody (CST, 48,453, 1:100) and CAV1 rabbit antibody (Abcam, ab32577, 1:250), followed by Alexa 647-conjugated goat anti-mouse antibody (Invitrogen, A32723, 1:500) and TRITC-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch, 111-025-003, 1:100) incubation. Colocalization was analyzed by Nikon NIS-Element AR Analysis software. Pearson’s correlation was used to quantify the degree of colocalization between fluorophores.

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Immunofluorescence Staining of Tumor and Animal Tissues

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Immunofluorescence staining was performed as previously described (Wu et al., 2019b (link)). Briefly, tumor and animal tissues were fixed with paraformaldehyde, dehydrated, and cleared with a gradient of alcohol solutions and xylene, respectively. Consecutively, tissues were embedded in paraffin. Paraffin-embedded tissues were sliced continuously (3.5°C) using a HistoCore AUTOCUT system (Leica), mounted, dewaxed, and rehydrated. For antigen retrieval, sections (3–4 μm) were pretreated in a microwave oven with citric acid buffer (pH 6.0) for 12 min and then cooled to 25°C in deionized water. After washing with PBS for 5 min, sections were incubated with 5% normal goat and donkey serum at 37°C for 1 h at 25°C and then incubated with the primary antibody (1:800, ab37647, Abcam) at 4°C overnight. After washing at least thrice with PBS, sections were incubated with the following secondary antibodies: Alexa 633-conjugated donkey anti-rat antibody (1:500, Invitrogen), Alexa 596-conjugated goat anti-rabbit (1:500, Invitrogen), and Alexa 488-conjugated goat anti-mouse antibody (1:250, Invitrogen) for 1 h at 25°C. Afterward, cells were extensively washed with PBS, and their nuclei were labeled using 4′,6-diamidino-2-phenylindole (DAPI). Free-floating sections with positive immunofluorescence staining were captured and analyzed using a laser scanning confocal microscope (Zeiss).

Wu K., Zeng J., Shi X., Xie J., Li Y., Zheng H., Peng G., Zhu G., Tang D, & Wu S. (2022). Targeting TIGIT Inhibits Bladder Cancer Metastasis Through Suppressing IL-32. Frontiers in Pharmacology, 12, 801493.

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Tau Oligomers Immunolabeling in SH-SY5Y Cells

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SH-SY5Y cells were treated with 2 μM tau oligomers or monomers for 6 h. Then, the cells on the coverslip were fixed and permeabilized. The samples were blocked for 1 h in 5% goat serum and incubated overnight at 4°C with Tau-5 (1:1000). Sections were then washed and incubated with an Alexa 488-conjugated goat anti-mouse antibody (1:700, Invitrogen) for 1 h at RT. Sections were then incubated overnight at 4°C with αAPF (1:350), then washed and incubated with an Alexa 568-conjugated goat anti-mouse antibody (1:700). DAPI was used to stain the nuclei (1:4000, Invitrogen). Sections were then washed for 30 min and coverslipped.

Lasagna-Reeves C.A., Sengupta U., Castillo-Carranza D., Gerson J.E., Guerrero-Munoz M., Troncoso J.C., Jackson G.R, & Kayed R. (2014). The formation of tau pore-like structures is prevalent and cell specific: possible implications for the disease phenotypes. Acta Neuropathologica Communications, 2, 56.

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Quantifying Amyloid-Beta and BACE1 in Lysosomes

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Cells were fixed with cold methanol (−20°C) for 10 min, washed with PBS, blocked with 5% goat serum, and incubated overnight at 4°C with primary antibodies including amyloid beta mouse antibody MOAB-2 (Biosensis, M-1586), BACE1 mouse antibody (Thermofisher, MA1–177) and lysosome-associated membrane protein-1 (LAMP1) rabbit antibody (Sigma, L1418). After washing with PBS, cells were incubated with fluorescence-conjugated secondary antibodies including Alexa 488-conjugated goat anti-mouse antibody (Invitrogen) and Alexa 546-conjugated goat anti-rabbit antibody (Invitrogen). Images were taken using our confocal microscope (Zeiss 800). Controls for specificity included staining with primary antibodies without fluorescence-conjugated secondary antibodies (background controls) and staining with only secondary antibodies; these controls eliminated auto-fluorescence in each channel and bleed-through (crossover) between channels. Averaged fluorescent intensities per cell was quantified using Image J software (NIH). Percentage of colocalization were quantified using Image J software (NIH) and data were normalized to control.

Hui L., Soliman M.L., Geiger N.H., Miller N.M., Afghah Z., Lakpa K.L., Chen X, & Geiger J.D. (2019). Acidifying endolysosomes prevented LDL-induced amyloidogenesis. Journal of Alzheimer's disease : JAD, 67(1), 393-410.

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