Alexa 488 goat anti rabbit igg

Manufactured by Thermo Fisher Scientific

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Alexa Fluor 488 goat anti-rabbit IgG is a secondary antibody conjugated with the Alexa Fluor 488 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassays and imaging applications.

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Alexa 488 (1863 citations) Alexa Fluor 488 goat anti-rabbit IgG (980 citations) Goat anti-rabbit Alexa 488 (232 citations) 488 goat anti-rabbit (9 citations) Goat anti-rabbit IgG-Alexa 488 conjugate (5 citations) Alexa Fluor 488 (green)-conjugated goat anti-rabbit IgG (5 citations) IgG Rabbit (4 citations) Alexa-488 anti-goat (3 citations) Alexa anti-rabbit 488 (3 citations) Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG (2 citations)

62 protocols using alexa 488 goat anti rabbit igg

Bacterial Expression and Purification of GST-RAP Fusion Protein

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GST and GST-RAP fusion protein were expressed in bacteria and then purified. Briefly, recombined plasmid was expressed in E. coli Transetta (DE3) cells. At OD₆₀₀ = 0.5, protein expression was induced with 1 mM IPTG (Solarbio) for 5 h at 25°C. Bacteria were lysed and sonicated in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 300 mM NaCl, 0.5% Triton X-100, PMSF, DTT, lysozyme, and protease inhibitor cocktail (Sigma-Aldrich) at 4°C. After centrifugation at 12,000 g for 15 min at 4°C, the supernatant was used for the protein purification with GST resin (TransGen Biotech) according to the manufacturer’s instructions. Elution of recombinant protein was performed under mild, non-denaturing condition using reduced glutathione. Then, the fractions were collected and analyzed with SDS-PAGE followed by Coomassie brilliant blue staining.
Breast cancer cells were treated with 200 nM GST-RAP fusion protein for 72 h followed by immunofluorescent staining. Briefly, cells grown on slides were fixed with 4% paraformaldehyde. After blocking, slides were stained with anti-GST antibody (Proteintech, 10000-0-AP). Slides were then incubated with Alexa-488 Goat anti-Rabbit IgG (Invitrogen, A11008). Nuclei were stained with DAPI (Thermo Fisher Scientific).

Yang M., Zhan Y., Hou Z., Wang C., Fan W., Guo T., Li Z., Fang L., Lv S., Li S., Gu C., Ye M., Qin H., Liu Q, & Cui X. (2022). VLDLR disturbs quiescence of breast cancer stem cells in a ligand-independent function. Frontiers in Oncology, 12, 887035.

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Immunocytochemistry of Primary Neurons

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For immunocytochemistry, coverslips with primary neurons were fixed with 4% paraformaldehyde/4% sucrose for 8 min, washed with PBS, and permeabilized with 0.3% Triton-X100/PBS for 10 min. After blocking in 5% NGS/PBS for 30 min, incubation with primary antibodies followed overnight at 4°C: rb-anti-pan-Synapsin (1:500, E028, T. Südhof, Stanford University), ms-anti-PSD-95 (1:500, NeuroMab), ch-anti-MAP2 (1:10,000, Abcam), rb-anti-Cofilin1 (1:7,000, Abcam), rb-anti-protein-interacting-with-C-kinase-1 (PICK1) (1:500, Proteintech), as well as Alexa568-phalloidin (1:100, Invitrogen), all diluted in 5% NGS/PBS. After washing, cells were incubated with the following secondary antibodies: Alexa488 goat-anti-rabbit IgG, Alexa647 goat-anti-chicken IgG (Invitrogen), Cy3-conjugated goat-anti-mouse IgG (Jackson Immuno Research), diluted 1:500 in 5% NGS/PBS for 1 h at RT. After additional washings in PBS, coverslips were embedded in mounting medium (Dako).

Blanque A., Repetto D., Rohlmann A., Brockhaus J., Duning K., Pavenstädt H., Wolff I, & Missler M. (2015). Deletion of KIBRA, protein expressed in kidney and brain, increases filopodial-like long dendritic spines in neocortical and hippocampal neurons in vivo and in vitro. Frontiers in Neuroanatomy, 9, 13.

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Antibody Validation for Protein Detection

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Mouse monoclonal anti-hnRNP K (D-6) antibody and rabbit polyclonal anti-nucleolin (H-250), normal mouse immunoglobulin G (IgG) and normal rabbit IgG antibodies were purchased from Santa Cruz Biotechnology. The rabbit polyclonal anti-human actin antibodies and rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibodies were purchased from Sigma. The mouse monoclonal HA antibody (MMS-101R) was purchased from Covance. The rabbit polyclonal anti-MOR antibodies (GTX10275), rabbit polyclonal anti-NeuN antibodies (GTX37604), mouse monoclonal Myc-tag antibody (9E10) and mouse monoclonal anti-α-tubulin antibody (GTX72360) were purchased from GeneTex. The rabbit polyclonal anti-Sp1 antibodies were purchased from Millipore. The Alexa-488 goat anti-rabbit IgG, Alexa-488 goat anti-mouse IgG, Alexa-568 goat anti-rabbit IgG and Alexa-568 goat anti-mouse IgG antibodies were purchased from Invitrogen.

Lee P.T., Chao P.K., Ou L.C., Chuang J.Y., Lin Y.C., Chen S.C., Chang H.F., Law P.Y., Loh H.H., Chao Y.S., Su T.P, & Yeh S.H. (2014). Morphine drives internal ribosome entry site-mediated hnRNP K translation in neurons through opioid receptor-dependent signaling. Nucleic Acids Research, 42(21), 13012-13025.

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Antibody Detection and Visualization Protocol

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The antibodies used in the present study were rabbit polyclonal antibody against rainbow trout GR (1:250) [29 (link)], Caveolin-1 mouse monoclonal antibody (1:500) [30 (link),31 (link)], ORAI-1 rabbit polyclonal antibody (1:250) [22 (link),32 (link),33 (link)], and phalloidin conjugated TRITC (1:500) for F-actin detection (1:500) [34 (link)]. The secondary antibodies used were Alexa 488 goat anti-rabbit IgG and Alexa 594 donkey anti-mouse IgG (Invitrogen, Burlington, ON, Canada).

Das C, & Vijayan M.M. (2023). Cortisol Rapidly Facilitates Glucocorticoid Receptor Translocation to the Plasma Membrane in Primary Trout Hepatocytes. Biology, 12(2), 311.

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Subcellular Localization of ATP11A

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36-48 hours after COS7 cells transfection, the transfected cells were washed twice with PBS, fixed with 4% paraformaldehyde, and washed three times with PBS. Cells were then permeabilized and blocked with normal goat serum, Triton X-100, and NaN₃ in PBS for 1 hour at room temperature. ATP11A protein was labeled with mouse anti-Flag antibody (1 : 2000, Sigma, Germany), and the endoplasmic reticulum (ER) was labeled with a rabbit anti-Calnexin antibody (1 : 1000, Cell Signaling Technology, CA, USA). The Golgi apparatus was labeled with a rabbit anti-GM130 antibody (1 : 1000, BD Biosciences, Mississauga, ON). The secondary antibodies used were Alexa 488 goat anti-rabbit IgG, Alexa 594 goat anti-mouse IgG (1 : 500, Invitrogen, USA). The images were captured under a Zeiss LSM 800 confocal scanning microscope.

Sun K., Tian W., Li X., Liu W., Yang Y, & Zhu X. (2020). Disease Mutation Study Identifies Critical Residues for Phosphatidylserine Flippase ATP11A. BioMed Research International, 2020, 7342817.

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Multicolor Flow Cytometry for Immune Cell Analysis

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Single-cell suspensions were stained using mouse-specific Abs, including FITC–anti-CD11b (BD Biosciences), Alexa 647–anti-IL-23p19 (eBioscience), PE–anti-TNF-α, PE/Cy7–anti-Ly6C for macrophages and subsets. For CD4 T cell and subset staining, cells were stained with FITC–anti-CD4, PE/Cy7–anti-Thy1.2, APC–anti-IFN-γ, and PE–anti-IL-17. Intracellular and intranuclear staining was performed as described previously (15 (link)). For macrophages treated with 2-D-gal in vitro, cells were stained with Bio-ICAM followed by streptavidin eFluor 450 (eBioscience) and APC–anti-CD11b, FITC–anti-CD80, and PE–anti-CD86. 2D2 CD4 T cells were identified by staining with APC–anti-CD4, PE/Cy7–Vβ11. DQ-Ovalbumin (DQ-OVA, Invitrogen) was detected in FITC channel. For macrophages loaded with Eα-GFP peptides, cells were stained with bio-YAe (eBioscience), followed by streptavidin eFluor 450 (eBioscience) and APC–anti-CD11b (Biolegend).
To determine the degree of ERK1/2 intracellular signaling, we performed phospho-flow studies according to the protocol from BD Biosciences. Intracellular pERK1/2 was stained with Rabbit anti-ERK1 (T202/Y204)/ERK2 (T185/Y187) (R&D systems), followed by Alexa488–goat anti-rabbit IgG (Invitrogen).
Data were acquired on a BD LSRII flow cytometer and analyzed using FlowJo software (Tree Star, Inc.).

Li J., Hsu H.C., Ding Y., Li H., Wu Q., Yang P., Luo B., Rowse A.L., Spalding D.M., Bridges SL J.r, & Mountz J.D. (2014). Inhibition of Fucosylation Reshapes Inflammatory Macrophages and Suppresses Type II Collagen-Induced Arthritis. Arthritis & rheumatology (Hoboken, N.J.), 66(9), 2368-2379.

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Immune Response Modulation Assay

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A647 anti-MICA/B, APC anti-CD3, FITC anti-CD56, APC anti-HLA-E, PE anti-HLA-A/B/C, blocking anti-NKG2D and all isotype control were from Biolegend. PE anti-MICA, -MICB, ULBP1, -ULBP2 and -ULBP3 were from R&D systems. FITC anti-pan γδTCR was from Beckman Coulter. PE anti-CD107a was from BD. Cetuximab (Erbitux) was from Merck. Recombinant human EGF was from Calbiochem. All cell culture media and reagents, Alexa 488 goat anti-rabbit IgG, and Prolongold were from Invitrogen. Chemical inhibitors and rabbit polyclonal anti-AUF1 and anti-phosphoAUF1 were from Sigma-Aldrich. The Dual Luciferase reporter system was from Promega.

Vantourout P., Willcox C., Turner A., Swanson C., Haque Y., Sobolev O., Grigoriadis A., Tutt A, & Hayday A. (2014). Immunological Visibility: Posttranscriptional Regulation of Human NKG2D Ligands by the EGF Receptor Pathway. Science translational medicine, 6(231), 231ra49.

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Whole-mount immunofluorescence staining of mouse embryos

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Mouse embryos were collected in phosphate-buffered saline (PBS) and directly immersed in 4% paraformaldehyde (PFA) in PBS (pH 7.4) at 4°C overnight. Following fixation, the samples were washed with PBS and stored in PBS at 4°C for further analyses. Back skins and hearts were dissected from fixed embryos and then stained by wholemount immunofluorescence staining as previously described (Yamazaki et al., 2017 (link)). The following primary antibodies were used in this study: polyclonal antibodies against NMHC 2A (1:1,000, Biolegend), NMHC2B (1:3,000, Biolegend), p-paxillin (1:1,000; BD Transduction Laboratories); monoclonal antibodies against PECAM1 (CD31, 1:200, BD Pharmingen), GFP (1:200, ab183734, Abcam). Fluorescence secondary antibodies used were Alexa 488 goat anti-rabbit IgG or Alexa 594 goat anti-mouse IgG (1:250, Invitrogen, Carlsbad, CA). Confocal microscopy was carried out either on a Leica TCS SP5 or on a Zeiss LSM 880. In all cases, when possible, comparison was made among littermates. For each genotype we analyzed at least three to five mice. We found no abnormalities in NM2A and NM2B single heterozygous mice or NM2A/2B double heterozygous mice compared with the littermates that expressed no Cre. Littermates containing no cre recombinase were used as controls for conditional ablated mice.

Ma X., Uchida Y., Wei T., Liu C., Adams R.H., Kubota Y., Gutkind J.S., Mukouyama Y.S, & Adelstein R.S. (2020). Nonmuscle myosin 2 regulates cortical stability during sprouting angiogenesis. Molecular Biology of the Cell, 31(18), 1974-1987.

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Immunostaining of Embryonic Eye Tissue

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Embryos were fixed overnight at 4°C in 4% PFA. Immunostaining of eyes was performed following standard procedures with minor modifications. For sections, samples were embedded in gelatin, cryo-sectioned at 10–12 μm, then the gelatin was melted in 42°C PBS. Wholemount anterior eye tissue or sections were washed in PBT then blocked in PBT containing 0.1% BSA and 5% heat-inactivated sheep serum. Samples were incubated overnight at 4°C in antibody solution containing rabbit anti-GFP (IgG, Invitrogen) at 1:500 or rat anti-endomucin (IgG2a, eBioscience) diluted 1:500 in blocking solution. After extensive washes in PBT, samples were blocked for one hour then incubated overnight at 4°C in secondary antibody (Alexa 488 goat anti-rabbit IgG, Invitrogen, or FITC goat anti-rat IgG2a, Bethyl) diluted 1/200 in blocking solution. Samples were washed in PBS. Some sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) to label all nuclei. All samples were mounted in PBS and imaged with a Zeiss AxioImager 2 fluorescence microscope with ApoTome and Axiocam (Carl Zeiss AG).

McKenna C.C., Ojeda A.F., Spurlin J I.I.I., Kwiatkowski S, & Lwigale P.Y. (2014). Sema3A maintains corneal avascularity during development by inhibiting Vegf induced angioblast migration. Developmental biology, 391(2), 241-250.

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Quantifying Immune Cell Infiltration in Lung and Kidney Tissues

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Lungs and kidneys were frozen in Tissue-Tek OCT compounds (Sakura, Tokyo, Japan) and cryosectioned at 5 µm thicknesses. The sections were fixed with cold acetone for 15 min and blocked with phosphate-buffered saline, 1% bovine serum albumin, and 1% goat serum albumin at room temperature for 1 h. Next, sections were incubated overnight at 4 °C with the following primary antibodies: 1 µg/mL rabbit anti-mouse CitH3 antibody (Abcam, Cambridge, UK) and 2 µg/mL rat anti-mouse Ly6G/C (neutrophil marker) antibody (Santa Cruz Biotechnology, Dallas, TX, USA). The sections were then washed and incubated with the following Alexa-conjugated secondary antibodies (Invitrogen): Alexa 488-goat anti-rabbit IgG (4 µg/mL) and Alexa 555-goat anti-rat IgG (4 µg/mL) at room temperature for 1 h. DNA was stained with 1 µg/mL TOPRO-3 (Invitrogen). Confocal images were acquired using a Leica TCS SP5 II microscope (Leica, Wetzlar, Germany). The percentages of CitH3-positive and Ly6G/C-positive areas in each field were calculated in 10 randomly selected non-overlapping fields from respective lung and kidney sections at ×400 magnification. The percentages of positive areas were averaged in respective organs and individual animals. The imaging analysis was conducted in a blinded manner using MCID software (MCIC, Linton, Cambridge, UK).

Hayase N., Doi K., Hiruma T., Matsuura R., Hamasaki Y., Noiri E., Nangaku M, & Morimura N. (2020). Recombinant thrombomodulin prevents acute lung injury induced by renal ischemia-reperfusion injury. Scientific Reports, 10, 289.

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