Alexa fluor 488 labeled goat anti rabbit igg antibody

Manufactured by Thermo Fisher Scientific

Sourced in United States

Alexa Fluor 488-labeled goat anti-rabbit IgG antibody is a secondary antibody conjugated with the Alexa Fluor 488 fluorescent dye. It is designed to bind to rabbit primary antibodies and can be used for various immunodetection techniques, such as immunofluorescence and Western blotting.

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Lab products found in correlation

Axio observer z1, by Zeiss (6 mentions) Alexa fluor 568 labeled goat anti guinea pig igg antibody, by Thermo Fisher Scientific (2 mentions) Zen software, by Zeiss (2 mentions) 48 well plate, by Greiner (1 mentions) Round bottom 96 well plate, by Greiner (1 mentions) Rabbit anti human irf5 antibody, by Cell Signaling Technology (1 mentions) Perm buffer 3, by BD (1 mentions) Canto 2, by BD (1 mentions) Lyse fix buffer, by BD (1 mentions) Metamorph image analysis software, by Molecular Devices (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions) Kaluza, by Beckman Coulter (1 mentions) Lsrfortessa, by BD (1 mentions) Axioplan 2 imaging system, by Zeiss (1 mentions) Lsm 900, by Zeiss (1 mentions) Leica tcs sp8 sted 3x, by Leica Microsystems (1 mentions) Plan apochromat, by Zeiss (1 mentions) Tmr red kit, by Roche (1 mentions) Bcip nbt, by Merck Group (1 mentions) Peroxidase conjugated anti dig antibody, by Roche (1 mentions)

10 protocols using alexa fluor 488 labeled goat anti rabbit igg antibody

Analysis of TBK1/IKKε Phosphorylation and IRF5 in DCs

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For analysis of TBK1/IKKε phosphorylation, DCs or macrophages were stimulated as indicated in 48-well plates (Greiner Bio-One) for 30 min and fixed using Lyse/Fix buffer (BD Biosciences) for 10 min at room temperature. For analysis of IRF5, unstimulated DCs were also lysed and transferred in a 96-well plate following the same protocol as TBK1 phosphorylation. Cells were harvested by gentle scraping, transferred to a 96-well round-bottom plate (Greiner Bio-One), washed in PBS, and permeabilized using Perm III buffer (BD Biosciences) for at least 30 min at −20°C. Cells were then washed in PBS containing 0.5% BSA and 0.1% sodium azide and stained for 1 h at RT with a rabbit-anti-human-IRF5 antibody (1:200) (Cell Signaling) or a rabbit-anti-human-pTBK1 antibody (1:50) (Ser172; Cell Signaling), which also reacts to pIKKε, followed by a 30 min staining at room temperature with Alexafluor488-labeled goat-anti-rabbit-IgG antibody (1:400) (Molecular Probes). Fluorescence was determined by flow cytometry (Canto II, BD Biosciences).

Hoepel W., Newling M., Vogelpoel L.T., Sritharan L., Hansen I.S., Kapsenberg M.L., Baeten D.L., Everts B, & den Dunnen J. (2019). FcγR-TLR Cross-Talk Enhances TNF Production by Human Monocyte-Derived DCs via IRF5-Dependent Gene Transcription and Glycolytic Reprogramming. Frontiers in Immunology, 10, 739.

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Quantifying NF-κB Activation in hiPS-RPE Cells

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hiPS-RPE cells (30,000–50,000) were seeded in 96 well plates. Cells were co-incubated with 1 μg/ml of LPS and 1 ng/ml of LCN2 for 24 h. Next day the culture medium was removed and cells were fixed by adding 100 μl of 4% formalin to the wells for 15 min, followed by washing in PBS twice for 5 min. Fixed cells were permeabilized with 100 μl of 0.2% Triton X-100 in PBS for 1 min at room temperature. The cells were then incubated with rabbit anti-mouse p65 (1:100 (eBioscience Ltd) in PBS containing 10% goat serum overnight at 4°C. Cells were then washed twice with PBS and incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG antibody (1:200) (Molecular Probes Inc.) in PBS at room temperature for 2 h. The cells were washed twice for 5 min with PBS. DAPI (1: 500; Vector Laboratories, UK) was added to the cells to visualize the nuclei. Cells were then washed twice with PBS and imaged under fluorescent microscope. Five high power fields (× 100 magnifications) were randomly selected in each sample for analysis. Positive nuclear staining in LPS treated cells was used as positive control for NF-κB staining. The percent of nuclear staining for NF-κB p65 was scored by counting the positive-stained cells and the total number of cells quantified in random microscopic fields using the Metamorph image analysis software (Molecular Devices, San Jose, CA).

Parmar T., Parmar V.M., Perusek L., Georges A., Takahashi M., Crabb J.W, & Maeda A. (2018). Lipocalin 2 plays an important role in regulating inflammation in retinal degeneration. Journal of immunology (Baltimore, Md. : 1950), 200(9), 3128-3141.

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Flow Cytometric Analysis of Bacteroides fragilis

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B. fragilis was grown to OD₆₀₀ = 0.5. 10 μl of the samples were added to 1 ml of PBS, and was washed twice with FACS buffer (2% fetal bovine serum, 1 mM EDTA, 0.1% sodium azide in PBS). PSB-specific antiserum was added and the sample was incubated for 1 h at 4°C. The sample was washed again twice with FACS buffer and incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG antibody (Molecular Probes) for 1 h at 4°C in the dark. The cells were washed with PBS and fixed with 4% of paraformaldehyde in the dark for 1h, following analysis by flow cytometry. Data were collected on LSR Fortessa (BD Biosciences) and analyzed with the ‘Kaluza’ software (Beckman Coulter). Bacteria cells positive for PSB were gated.

Ben-Assa N., Coyne M.J., Fomenkov A., Livny J., Robins W.P., Muniesa M., Carey V., Carasso S., Gefen T., Jofre J., Roberts R.J., Comstock L.E, & Geva-Zatorsky N. (2020). Analysis of a phase-variable restriction modification system of the human gut symbiont Bacteroides fragilis. Nucleic Acids Research, 48(19), 11040-11053.

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Immunofluorescence Labeling of Bacteria

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Bacteria were grown in basal media to OD₆₀₀ of 0.6, washed, and incubated with a rabbit antiserum specific to PSB (1 (link)). Cells were washed and mixed with Alexa Fluor 488-labeled goat anti-rabbit IgG antibody (Molecular Probes). Bacteria were washed and imaged using an Axioplan 2 imaging system (Zeiss) at 600×.

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Immunofluorescent Detection of P2Y6R in Liver

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For tissue sections, the liver tissue was fixed by 4% paraformaldehyde. Frozen sections (5 μm thick) were cut and prepared for immunofluorescent staining [67 (link)]. The expression of P2Y₆R was detected using rabbit anti-P2Y₆R antibodies (#APR011: Alomone Labs, Jerusalem, Israel). The immunoreactivity of P2Y₆R was detected using an Alexa Fluor 488-labeled goat anti-rabbit IgG antibody (#A-11008: ThermoFisher Scientific, Waltham, MA, USA). Non-specific immunoreactivity was blocked with 10% normal goat serum, 1% BSA, and 0.3% Triton X-100 in PBS. After incubation with the secondary antibody, images were captured using a confocal laser-scanning microscope (LSM900, Zeiss, Oberkochen, Germany).

Nishiyama K., Ariyoshi K., Nishimura A., Kato Y., Mi X., Kurose H., Kim S.G, & Nishida M. (2023). Knockout of Purinergic P2Y6 Receptor Fails to Improve Liver Injury and Inflammation in Non-Alcoholic Steatohepatitis. International Journal of Molecular Sciences, 24(4), 3800.

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Immunofluorescence Staining of CD9 Protein

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Cells were grown on coverslips in media and were fixed with 4% paraformaldehyde for 10 min. Then, the cells were washed thrice with PBS, permeabilized with 0.1% Triton X-100 for 15–20 min, and again washed thrice. Cells were then treated with 1% BSA in PBS (IH solution) for 45 min followed by incubation with anti-CD9 rabbit monoclonal antibody (1 μg/mL) in IH solution at 4 °C overnight. Then, the samples were thoroughly washed thrice with PBS and exposed to Alexa Fluor 488-labeled goat anti-rabbit IgG antibody (Thermo Scientific, USA). Fluorescence was measured using a laser scanning confocal microscope (Leica TCS SP8 STED 3X, Leica Microsystems, Germany).

Thapa R.K., Nguyen H.T., Jeong J.H., Kim J.R., Choi H.G., Yong C.S, & Kim J.O. (2017). Progressive slowdown/prevention of cellular senescence by CD9-targeted delivery of rapamycin using lactose-wrapped calcium carbonate nanoparticles. Scientific Reports, 7, 43299.

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Immunofluorescence Staining of PRRSV Proteins

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Cells were fixed with cold methanol-acetone (1:1) for 15 min at 4°C and washed with phosphate-buffered saline (PBS) three times. After blocking with 1% BSA–PBS for 30 min at room temperature, the cells were incubated with antibodies against pCD163 (mAb 2A10, AbD Serotec, and rabbit anti- pCD163 polyclonal antibody, produced by Genscript Company, CHINA), or pCD169 (rabbit anti-pCD169 polyclonal antibody, produced by Genscript Company, CHINA) or sCD151 (mAb 11G5a, AbD Serotec) at 37°C for 1 h. After washing with PBS three times, the cells were incubated with an Alexa Fluor 488-labeled goat anti-rabbit IgG antibody (Invitrogen, Cat. A-11008) or Alexa Fluor 594-labeled goat anti-mouse IgG antibody (Invitrogen, Cat. A-11005) at 37°C for 1 h. Finally, cells were counterstained with DAPI and examined using a fluorescence microscope. For detection of PRRSV infection in transgenic BHK-21 cells and MARC-145 cells, the PRRSV N protein was stained with SDOW17 (mAb; Rural Technologies, Cat. HB-10997) and observed using Alexa Fluor 594-labeled goat anti-mouse IgG antibody staining.

Zhang L., Cui Z., Zhou L., Kang Y., Li L., Li J., Dai Y., Yu S, & Li N. (2016). Developing a Triple Transgenic Cell Line for High-Efficiency Porcine Reproductive and Respiratory Syndrome Virus Infection. PLoS ONE, 11(5), e0154238.

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Immunostaining of Cells for Confocal Imaging

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Immunostaining of cells was performed as described (Okumoto et al., 2011 (link)) with 4% paraformaldehyde for cell fixation and 0.1% Triton X-100 for permeabilization. Immuno-complexes were visualized with an Alexa Fluor 488-labeled goat anti-rabbit IgG antibody and an Alexa Fluor 568-labeled goat anti-guinea pig IgG antibody (Invitrogen). Cells were observed by a confocal laser microscope (LSM710 with Axio Observer Z1; Zeiss) equipped with a Plan Apochromat 100 × 1.4 NA oil immersion objective lens and argon plus dual HeNe lasers at RT. Images were acquired with Zen software (Zeiss) and prepared using Photoshop (CS4; Adobe).

Okumoto K., El Shermely M., Natsui M., Kosako H., Natsuyama R., Marutani T, & Fujiki Y. (2020). The peroxisome counteracts oxidative stresses by suppressing catalase import via Pex14 phosphorylation. eLife, 9, e55896.

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Dual-labeling of cmyb RNA and pH3

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Antisense RNA probes were transcribed using linearized constructs with T3 or T7 polymerase (Ambion) in the presence of digoxigenin (DIG, Roche)-labeled UTP using the DIG-RNA Labeling Kit (Roche). WISH was performed as described previously using NBT/BCIP (Sigma) as substrates^{76 (link),77 (link)}. TUNEL was performed with In Situ Cell Death Detection Kit and TMR Red Kit (Roche) following manufacturer's instruction. To detect both cmyb RNA and mitosis marker pH3 simultaneously, embryos were first hybridized with the DIG-labeled antisense cmyb RNA probe, incubated at 4 °C overnight with a peroxidase-conjugated anti-DIG antibody (1:500; Roche), and stained with Alexa Fluor cy3-conjugated tyramide as substrate (PerkinElmer). The embryos were then incubated with primary anti-pH3 (ser10) antibody (1:500; Santa Cruz), and finally incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG antibody (1:500; Invitrogen).

Jia X.E., Ma K., Xu T., Gao L., Wu S., Fu C., Zhang W., Wang Z., Liu K., Dong M., Jing C., Ren C., Dong Z., Chen Y., Jin Y., Huang Q., Chang X., Deng M., Li L., Luo L., Zhu J., Dang Y., Chang H.C., Zon L.I., Zhou Y., Chen S, & Pan W. (2015). Mutation of kri1l causes definitive hematopoiesis failure via PERK-dependent excessive autophagy induction. Cell Research, 25(8), 946-962.

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Immunostaining Visualization by Confocal Microscopy

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Immunostaining of cells was performed as described (Okumoto et al., 2011) with 4% paraformaldehyde for cell fixation and 0.1% Triton X-100 for permeabilization.
Immuno-complexes were visualized with Alexa Fluor 488-labeled goat anti-rabbit IgG antibody and Alexa Fluor 568-labeled goat anti-guinea pig IgG antibody (Invitrogen). Cells were observed by a confocal laser microscope (LSM710 with Axio Observer Z1; Zeiss) equipped with a Plan Apochromat 100x 1.4 NA oil immersion objective lens and argon plus dual HeNe lasers at RT. Images were acquired with Zen software (Zeiss) and prepared using Photoshop (CS4; Adobe).

Okumoto K., Shermely M.E., Natsui M., Kosako H., Natsuyama R., Marutani T., & Fujiki Y. (2020). Peroxisome counteracts oxidative stresses by suppressing catalase import via Pex14 phosphorylation.

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