Alexa 488 conjugated goat anti rabbit igg

Manufactured by Thermo Fisher Scientific

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Alexa Fluor 488-conjugated goat anti-rabbit IgG is a secondary antibody used for detecting and visualizing rabbit primary antibodies in various immunoassays and imaging applications. The Alexa Fluor 488 dye provides bright, photostable fluorescence for sensitive detection.

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113 protocols using alexa 488 conjugated goat anti rabbit igg

DRG Immunofluorescence Labeling Protocol

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Ten L3 and L4 DRG samples were harvested from the 9-month-old C57BL/6J and STR/Ort mice and were then immersed in a buffered paraformaldehyde fixative at 4°C overnight. The samples were further incubated in PBS containing 20% sucrose for 24 h at 4°C. After freezing in liquid nitrogen, each specimen was sectioned at 10 μm thickness on a cryostat (Leica Microsystems, CM3050S, Wetzlar, Germany) and was then treated for 90 min at room temperature with a blocking solution of PBS containing 0.05% Tween-20 and 1% skim milk.
The sectioned DRG specimens were stained with rabbit antibodies against calcitonin gene-related peptide (CGRP; 1 : 1000; Immunostar, Hudson, WI, USA) and transient receptor potential vanilloid 1 (TRPV1; 1 : 500; Calbiochem, San Diego, CA, USA) by incubation for 18 h at 4°C. The DRG sections were then incubated with Alexa 488-conjugated goat anti-rabbit IgG (for CGRP immunoreactivity, 1 : 1000; Molecular Probes) and Alexa 488-conjugated goat anti-rabbit IgG (for TRPV1 immunoreactivity, 1 : 1000; Molecular Probes). The immunostained sections were visualized using a fluorescence microscope (Axiovert 200, Zeiss, Jena, Germany) in a treatment-blinded manner. The numbers of CGRP- and TRPV-positive cells were counted, and the proportion of these cells to the total number of nucleated cells in DRG was calculated for each DRG sample.

Takano S., Uchida K., Miyagi M., Inoue G., Fujimaki H., Aikawa J., Iwase D., Minatani A., Iwabuchi K, & Takaso M. (2016). Nerve Growth Factor Regulation by TNF-α and IL-1β in Synovial Macrophages and Fibroblasts in Osteoarthritic Mice. Journal of Immunology Research, 2016, 5706359.

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Caco-2 Cell Immunostaining for NHE3 and Rab

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Caco-2bbe/NHE3 cells grown on Transwells (Corning, Lowell, MA) were fixed using 100% methanol at −20°C for 20 minutes. For NHE3 and apical membrane staining, cells were washed three times with PBS, incubated with WGA, Alexa Flour 647-conjugate for 10 minutes, permeabilized using 0.05% Triton X-100 in PBS, and blocked with 5% normal goat serum for 1 hour at RT. Cells were then stained with rabbit anti-VSVG antibody for 16 hours at 4°C. Cells were washed with PBS three times and incubated with Alexa 488-conjugated goat anti-rabbit IgG (Invitrogen) for 1 hour at RT. After three 10 minutes washes with PBS, specimens were mounted with ProLong Glass Antifade Reagent (Invitrogen) and observed under a Nikon A1R HD confocal microscope (Nikon Instruments Inc., NY) coupled to a Plan Apo λ 60x Oli lens. For NHE3 and Rab staining, cells were stained with rabbit anti-VSVG and mouse anti-Rab5a, or mouse anti-Rab7 antibody for 16 hours at 4 C. After three washes for 10 minutes each with PBS, cells were incubated with Alexa 488-conjugated goat anti-rabbit IgG and Alexa 568-conjugated goat anti-mouse IgG (Invitrogen) for 1 hour at room temperature. Pearson’s correlations of NHE3 and Rab were determined using NIS-Elements AR software (Nikon).

Han Y, & Yun C.C. (2020). Ubiquitin-specific peptidase 7 (USP7) and USP10 mediate deubiquitination of human NHE3 regulating its expression and activity. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(12), 16476-16488.

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Visualizing TRAIL Expression in MDR-Off Cells

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HeLa MDR-Off cells in the absence and presence of doxycycline and KB-C1 shABCB1 and shNS were immunolabeled with primary antibody TRAIL at 1:100 and then with secondary antibody Alexa 488-conjugated goat anti-rabbit IgG at 1:500 (Molecular Probes, Grand Island, NY). The procedures were performed as previously described [22 (link)] and cell staining was visualized in a Zeiss LSM 510 NLO Meta confocal microscope.

Souza P.S., Madigan J.P., Gillet J.P., Kapoor K., Ambudkar S.V., Maia R.C., Gottesman M.M, & Fung K.L. (2015). Expression of the multidrug transporter P-glycoprotein is inversely related to that of apoptosis-associated endogenous TRAIL. Experimental cell research, 336(2), 318-328.

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Subcellular Localization of Hsp90-Survivin Complexes

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Heat shock protein 90 and LDL were conjugated with Alexa Fluor 594 (Molecular Probes) according to the manufacturer's instructions. Monocyte-derived DCs were incubated at 37°C with Alexa Fluor 594-labeled Hsp90 (20 μg) complexed with survivin-2B_75-93 peptide (20 μg) for 1 h. Following incubation, cells were washed twice with ice-cold PBS and fixed with ice-cold acetone for 1 min. Organelles were stained with an anti-Rab5 pAb and EEA1 mAb for early endosomes and anti-LAMP-1 pAb for late endosomes followed by Alexa 488-conjugated goat anti-rabbit IgG (Molecular Probes) or anti-mouse IgG (Molecular Probes) and then visualized with a Bio-Rad MRC1024ES confocal scanning laser microscope system (Bio-Rad, Richmond, CA, USA). For evaluation of colocalization, a single z-plane of one cell was evaluated. For each protein and organelle combination, a total of 150 cells (50 cells from three independent experiments) were analyzed.

Tanaka T., Okuya K., Kutomi G., Takaya A., Kajiwara T., Kanaseki T., Tsukahara T., Hirohashi Y., Torigoe T., Hirata K., Okamoto Y., Sato N, & Tamura Y. (2014). Heat shock protein 90 targets a chaperoned peptide to the static early endosome for efficient cross-presentation by human dendritic cells. Cancer Science, 106(1), 18-24.

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Immunofluorescence Staining of Foxo3a

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Cells grown on coverslips were fixed in 4% paraformandehyde in PBS for 10 mins at 4℃, permeablized in 0.25% Triton X-100 in PBS for 10 mins, and blocked in 5% donkey serum (Chemicon) and 0.3% Triton X-100 in PBS for 30 mins at RT. Next, cells were incubated with rabbit polyclonal anti-Foxo3a antibody (Cell signaling) (1:200) in 1% BSA and 0.3% Trition X-100 in PBS for overnight at 4℃. After that, incubation of cells with Alexa 488-conjugated goat anti-rabbit IgG (Molecular Probes) (1:1000) in 1% BSA and 0.3% Trition in PBS was carried out in RT. After deeply washing the cells with PBS, DAPI (1 µg/ml in PBS) was added to stain nuclei. Cells were mounted with Fluoromount-G (Southern biotech) and images were taken by a fluorescence microscope.

Nguyen L.T., Lee Y.H., Sharma A.R., Park J.B., Jagga S., Sharma G., Lee S.S, & Nam J.S. (2017). Quercetin induces apoptosis and cell cycle arrest in triple-negative breast cancer cells through modulation of Foxo3a activity. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 21(2), 205-213.

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Immunohistochemical Profiling of Neural Markers

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A mouse monoclonal antibody against GM2 ganglioside (GMB28; immunoglobulin M, 1:20) was kindly donated by Dr. Tai (Department of Tumor Immunity, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). An anti-GFAP (Dako, Carpinteria, CA, USA, 1:1000), anti-Iba1 (Wako Pure Chemical Industries, Osaka, Japan, 1:500), anti-CD68 (clone FA-11, AbD Serotec Ltd., Oxford, UK, 1:100), anti-NeuN (EMD Millipore, Billerica, MA, 1;1000) and anti-S100ß (GeneTex, Irvine, CA, 1:100) were used as primary antibodies. As secondary antibodies, Alexa-488-conjugated goat anti-mouse IgG (1:1000), Alexa-568-conjugated goat anti-mouse IgG (1:1000), Alexa-568-conjugated goat anti-mouse IgM (1:1000), Alexa-488-conjugated goat anti-rat IgG (1:1000), Alexa-488-conjugated goat anti-rabbit IgG (1:1000), Alexa-568-conjugated goat anti-rabbit IgG (1:1000) (all purchased from Molecular Probes, Eugene, OR, USA), and Histofine Simple Stain MAX-PO(R) (Nichirei Co., Tokyo, Japan) were used.

Ogawa Y., Sano T., Irisa M., Kodama T., Saito T., Furusawa E., Kaizu K., Yanagi Y., Tsukimura T., Togawa T., Yamanaka S., Itoh K., Sakuraba H, & Oishi K. (2017). FcRγ-dependent immune activation initiates astrogliosis during the asymptomatic phase of Sandhoff disease model mice. Scientific Reports, 7, 40518.

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Multimodal Histological Characterization

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Bielschowsky silver impregnation and Luxol fast blue periodic acid Schiff (LFB‐PAS) staining were performed according to standard procedures. Primary antibodies utilized for immunohistochemistry were against injured axons (APP, 1:2,000, clone 22C11, Chemicon) and nonphosphorylated neurofilaments (SMI32, 1:1,000, Covance, Princeton, NJ, USA), healthy phosphorylated neurofilaments/axons (SMI31, 1:10,000, Covance, Princeton, NJ, USA), myelin basic protein (MBP, 1:2,000, Dako), activated microglia (Mac3, also known as Lamp2, 1:200, clone M3/84, BD Pharmingen), glial fibrillary acidic protein (GFAP, 1:1,000, Dako), and foamy monocytes and macrophages (CD68, 1:5,000, clone KiM1P). Biotinylated secondary antibodies (GE Healthcare, Jackson ImmunoResearch and DCS Innovative diagnostic system), peroxidase conjugated avidin and DAB (Sigma‐Aldrich) were used for immunohistochemistry. Fluorescence labeled secondary antibodies (Cy3‐conjugated goat anti‐mouse IgG, 1:200, Jackson ImmunoResearch and Alexa488‐conjugated goat anti‐rabbit IgG, 1:200, Molecular Probes, Life technologies) were used for fluorescence immunohistochemistry.

Schultz V., van der Meer F., Wrzos C., Scheidt U., Bahn E., Stadelmann C., Brück W, & Junker A. (2017). Acutely damaged axons are remyelinated in multiple sclerosis and experimental models of demyelination. Glia, 65(8), 1350-1360.

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Immunofluorescence Staining of Cellular Proteins

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Cells were plated on coverslips, and after 24 h of co-culturing, cells were fixed with 4% paraformaldehyde for 20 min and incubated with 10 mM NH₄Cl for 10 min. The subsequent procedures were performed as previously described.8 (link) We used anti-Pgp (clone UIC2; Coulter), anti-Yb-1 (Abcam) and anti-NF-κB primary antibodies and Alexa 488-conjugated goat anti-rabbit IgG or Alexa 594-conjugated goat anti-mouse IgG secondary antibodies (Molecular Probes, Eugene, OR, USA). Images were acquired with the NIS-Elements F2.30 software, using an Eclipse E200 Nikon microscope connected to a Digital Sight system.

de Souza P.S., Cruz A.L., Viola J.P, & Maia R.C. (2014). Microparticles induce multifactorial resistance through oncogenic pathways independently of cancer cell type. Cancer Science, 106(1), 60-68.

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Immunofluorescence Analysis of Skin Markers

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Immunofluorescence staining was performed on 20-µm sections using polyclonal rabbit-anti-Ki67 (1:400, ab15580, Abcam), monoclonal rabbit anti-K17 (1:200, #4543, Cell Signaling Technology), monoclonal anti-K14 (1:200, ab7880, Abcam), monoclonal rabbit anti-phospho-Stat3 (pStat3, 1:200, #9145, Cell Signaling Technology), monoclonal rabbit anti-Stat3 (1:200, #9139, Cell Signaling Technology) overnight at 4 °C. Then, Alexa488-conjugated goat-anti-rabbit IgG (1:400, A21206, Molecular Probes) or Alexa546-conjugated goat-anti-mouse IgG (1:400, A11003, Molecular Probes) was used as secondary antibody. The sections were then counterstained with DAPI (1:500, Dojindo) and mounted with fluorescent mounting medium (Dako). At least three embryos of each genotype were used for each analysis.

Sarper S.E., Kurosaka H., Inubushi T., Ono Minagi H., Kuremoto K.I., Sakai T., Taniuchi I, & Yamashiro T. (2018). Runx1-Stat3-Tgfb3 signaling network regulating the anterior palatal development. Scientific Reports, 8, 11208.

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Quantifying Proliferative Cells in Palate Development

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Immunofluorescence staining was performed using polyclonal rabbit-anti-Ki67 (1:400, ab15580, Abcam), polyclonal rabbit anti-K6 (1:200, #4543, 905701, Biolegend), monoclonal anti-K14 (1:200, ab7880, Abcam), monoclonal rabbit anti-phospho-Stat3 (pStat3, 1:200, #9145, Cell Signaling Technology) or monoclonal rabbit anti-Stat3 (1:200, #9139, Cell Signaling Technology) overnight at 4°C. Alexa488-conjugated goat-anti-rabbit IgG (1:400, A21206, Molecular Probes) or Alexa546-conjugated goat-anti-mouse IgG (1:400, A11003, Molecular Probes) was used as secondary antibody. DAPI (1:500, Dojindo) was used for nuclear staining and the sections were mounted with fluorescence mounting medium (Dako). At least three embryos of each genotype were used for each analysis.
The percentage of proliferating cells at the fusing or contacting epithelium between the primary and the secondary palate was determined by counting Ki67-positive cells and reporting this value as a percentage of the total number of cells, as determined by DAPI staining.

Sarper S.E., Inubushi T., Kurosaka H., Ono Minagi H., Murata Y., Kuremoto K.I., Sakai T., Taniuchi I, & Yamashiro T. (2019). Anterior cleft palate due to Cbfb deficiency and its rescue by folic acid. Disease Models & Mechanisms, 12(6), dmm038851.

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