Alexa fluor 568 donkey anti rabbit igg

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

Alexa Fluor 568 donkey anti-rabbit IgG is a secondary antibody conjugated with the Alexa Fluor 568 fluorescent dye. It is used to detect and visualize rabbit primary antibodies in various immunoassays and imaging techniques.

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Alexa Fluor-568-conjugated donkey anti-rabbit IgG Alexa Fluor 568-labeled donkey anti-rabbit IgG Alexa Fluor 568 donkey anti-rabbit IgG (H + L) Alexa Fluor® 568 conjugated donkey anti-Rabbit IgG (H+L) secondary antibody Alexa Fluor 568 donkey anti-rabbit IgG antibody Alexa Fluor 568 donkey anti-rabbit Alexa Fluor® donkey anti-rabbit IgG Donkey anti-rabbit Alexa 568 Alexa Fluor 568 anti-rabbit Alexa Fluor 568 IgG

54 protocols using alexa fluor 568 donkey anti rabbit igg

Immunofluorescence Assay for FUS Localization

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SH-SY5Y cells were seeded on gelatin-treated glass coverslips and transfected with EGFP-tagged FUS constructs [32 ]. Next day, the cells were fixed in 4% (w/v) paraformaldehyde in 1x PBS, permeabilized with 0.25% (v/v) Triton-X100 in 1x PBS and blocked with 10% (w/v) bovine serum albumin (BSA) in 1x PBS. The slides were incubated with primary antibodies diluted in 3% (w/v) BSA/1x PBS. The primary antibodies were the same as those used for immunoblotting. The secondary antibodies were Alexa Fluor 568 Donkey Anti-Rabbit IgG or Alexa Fluor 568 Donkey Anti-Mouse IgG (Life Technologies, A10042 and A10037, respectively). The nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI). The coverslips were mounted on glass slides with Vectashield Mounting Medium (Vector Laboratories, H-1000-10). Confocal microscopic images were acquired using a Nikon A1 confocal microscope with a 60x objective.

Kamelgarn M., Chen J., Kuang L., Arenas A., Zhai J., Zhu H, & Gal J. (2016). Proteomic analysis of FUS interacting proteins provides insights into FUS function and its role in ALS. Biochimica et biophysica acta, 1862(10), 2004-2014.

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Cerebellar Immunostaining in Marmoset

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Five weeks after the viral injection, the marmoset was anesthetized as described above and perfused intracardially with PBS followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The cerebellum was subsequently dissected, and 100-μm-thick sections were cut and immunostained using the same protocol as in mice. Primary antibodies used were rat monoclonal anti-GFP (1:1,000; 04404-84; Nacalai Tesque) and rabbit polyclonal anti-Parvalbumin (1:200: PV-Rb-Af750; Frontier Institute), whereas the secondary antibodies were Alexa Fluor 488 donkey anti-rat IgG (1:1,000; Life Technologies) and Alexa Fluor 568 donkey anti-rabbit IgG (1:1,000; Life Technologies).

Nitta K., Matsuzaki Y., Konno A, & Hirai H. (2017). Minimal Purkinje Cell-Specific PCP2/L7 Promoter Virally Available for Rodents and Non-human Primates. Molecular Therapy. Methods & Clinical Development, 6, 159-170.

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Quantification of Histone Modifications

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Immunostaining, fluorescence detection and quantification were described previously^{33 (link)}. Primary antibodies were mouse anti-H3K27me3 (1:500, Active Motif, 61017) and rabbit anti-H2Aub (1:2,000, Cell Signaling, 8240). Secondary antibodies were Alexa Fluor 488 donkey anti-mouse IgG (1:200) or Alexa Fluor 568 donkey anti-rabbit IgG (1:200) (Life Technologies).

Chen Z., Djekidel M.N, & Zhang Y. (2021). Distinct dynamics and functions of H2AK119ub1 and H3K27me3 in mouse preimplantation embryos. Nature genetics, 53(4), 551-563.

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Quantitative EGFR Phosphorylation Assay

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Primary antibodies: Mouse monoclonal antibody PY72 (Glenney et al., 1988 (link)) (InVivo Biotech Services, Henningsdorf, Germany), rabbit anti EGFR pY₁₀₆₈ (Cell Signaling; 1:400), goat anti EGFR (R&D Systems; 1:300). Secondary antibodies: Alexa Fluor® 568 donkey anti-rabbit IgG (Life Technologies, 1:200), Alexa Fluor® 568 donkey anti-mouse IgG (Life Technologies, 1:200), Alexa Fluor® 647 donkey anti-goat IgG (Life Technologies, 1:200), Alexa Fluor® 647 chicken anti-mouse IgG (Life Technologies, 1:200), Alexa Fluor® 647 donkey anti-rabbit IgG (Life Technologies, 1:200), IRDye® 800CW Donkey anti-Rabbit IgG (Licor, 1:10000), IRDye® 680RD Donkey anti-Mouse IgG (Licor, 1:10000).

Stanoev A., Mhamane A., Schuermann K.C., Grecco H.E., Stallaert W., Baumdick M., Brüggemann Y., Joshi M.S., Roda-Navarro P., Fengler S., Stockert R., Roßmannek L., Luig J., Koseska A, & Bastiaens P.I. (2018). Interdependence between EGFR and Phosphatases Spatially Established by Vesicular Dynamics Generates a Growth Factor Sensing and Responding Network. Cell Systems, 7(3), 295-309.e11.

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Detecting Lung Cell Labeling

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Animals were euthanized by an intraperitoneal injection of 150 mg/kg Lethobarb. The total lung tissue was excised following perfusion with 4% paraformaldehyde (PFA) via tracheal intubation. Total lung tissue was ligated at the upper trachea, excised and immersed in 4% PFA for 48 h and processed for histology. To detect labeled cells histologically, sections from throughout the total lung were cut and incubated with primary antibody for mKATE (1:500 rabbit polyclonal Anti-tRFP antibody, #AB234, Evrogen, Farmingdale, NY) for 1 h at room temperature. Sections were then incubated with secondary antibody (AlexaFluor^® 568 donkey anti-rabbit IgG, Life Technologies, Grand Island, NY) for 1 h at room temperature. Slides were counter stained with DAPI (Sigma, St. Louis, MO) and mounted with Dako Fluorescent Mounting Medium (Dako, Carpinteria, CA). Slides were visualized with the Leica DM4000B fluorescent microscope to determine presence of labeled cells.

Murphy S.V., Hale A., Reid T., Olson J., Kidiyoor A., Tan J., Zhou Z., Jackson J, & Atala A. (2015). Use of trimetasphere metallofullerene MRI contrast agent for the non-invasive longitudinal tracking of stem cells in the lung. Methods (San Diego, Calif.), 99, 99-111.

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Deciphering Immune Response Pathways

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Anti-CD11c (N418), MHC class II (I-A) (NIMR-4), anti-CD40 (HM40-3), Anti-CD80 (16-10A1), Anti-CD86 (GL1) and anti-mTLR9 (M9.D6) were obtained from eBioscience. Anti-β-actin (AC-74) and thioglycolate were from Sigma-Aldrich. Anti-p44/42 MAPK (4695), anti-phospho-p44/42 MAPK (9101), anti-p38 MAPK (9212), anti-phospho-p38 MAPK (9216), anti-IkB-α (9242), anti-phospho-IkBα (2859), anti-SAPK/JNK (9258), anti-HA (2367 and 3724) and anti-phospho-SAPK/JNK (9255) were from Cell Signaling Technology. Anti-EEA1 was from Thermo Fisher Scientific. Anti-LAMP1 (1D4B) was from abcam. Alexa Fluor488 Donkey anti-mouse IgG, Alexa Fluor488 Donkey anti-rat IgG and Alexa Fluor568 Donkey anti-rabbit IgG were from life technologies. Affinity-purified rabbit polyclonal anti-CRAMP Ab which recognizes CRAMP peptide domain was previously generated in our laboratory(38 (link)). ODN 1585, ODN 1668, ODN M362, ODN 1668-FITC, ODN M362-FITC, R848, Poly(I:C) HMW, Pam3CSK4, Zymozan, LPS-EB, pUNO-mTLR9-HA and pSELECT-puro-mcs were from invivogen. DOTAP liposomal transfection reagent (DOTAP) was from Roche applied science. Recombinant mouse CRAMP peptide (mCRAMP) was synthesized by Genemed Synthesis Inc.

Nakagawa Y, & Gallo R.L. (2014). Endogenous intracellular cathelicidin enhances TLR9 activation in dendritic cells and macrophages. Journal of immunology (Baltimore, Md. : 1950), 194(3), 1274-1284.

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Immunostaining of Transgenic Embryos

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The embryos were fixed with 3.7% paraformaldehyde (PFA) in PBS at room temperature for 20 min, and then washed with PBS containing 0.1 mg/mL bovine serum albumin (BSA; Sigma-Aldrich) (0.1% BSA in PBS) three times. The membrane was permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) in PBS (0.5% Triton X-100 in PBS) at room temperature for 15 min. After being washed three times with 0.1% BSA in PBS, the embryos were treated with the primary antibody against RFP (Abcam, Cambridge, UK; diluted 500-fold with 0.1% BSA in PBS) overnight at 4 °C followed by treatment with the secondary antibody (AlexaFluor 568 donkey-anti rabbit IgG; Life Technologies; diluted 200-fold with 0.1% BSA in PBS) at room temperature for 1 h. After being washed with 0.1% BSA in PBS three times, the samples were mounted in VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA, USA) containing 3 µg/mL 4,6-diamidino-2-phenylindole (DAPI: Dojindo Laboratories, Kumamoto, Japan) on a glass slide. The fluorescence was observed using an FV3000 confocal laser-scanning microscope (Olympus, Tokyo, Japan).

Sugie K., Funaya S., Kawamura M., Nakamura T., Suzuki M.G, & Aoki F. (2020). Expression of Dux family genes in early preimplantation embryos. Scientific Reports, 10, 19396.

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Fluorescent Secondary Antibody Protocol

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IRDye 680 donkey anti‐mouse IgG, IRDye 800 donkey anti‐rabbit IgG, (LI‐COR Biosciences, Lincoln, NE); Alexa Fluor 647 chicken anti‐rabbit IgG, Alexa Fluor 647 donkey anti‐goat IgG, Alexa Fluor 568 donkey anti‐rabbit IgG (Life Technologies, Darmstadt, Germany).

Joshi M.S., Stanoev A., Huebinger J., Soetje B., Zorina V., Roßmannek L., Michel K., Müller S.A, & Bastiaens P.I. (2023). The EGFR phosphatase RPTPγ is a redox‐regulated suppressor of promigratory signaling. The EMBO Journal, 42(10), e111806.

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Immunofluorescence Staining on Tissue Sections

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Histology on cryosections and paraffin sections was performed using standard methods as detailed in SI Appendix, SI Materials and Methods.
The following antibodies or labeling agents were used for immunostainings: rat anti-CD45 (30F11; eBiosciences), goat anti-mFSTL1 (AF1738; R&D Systems), goat anti-mouse PDGFRα (AF1062; R&D Systems), phalloidin Alexa Fluor 488 (A12379; Life Technologies), phalloidin Atto 647N (Sigma), rabbit anti-RFP/tdTomato (600–401-379; Rockland), mouse anti-Ki67 (556003; BD Biosciences), Alexa Fluor 568 donkey anti-rabbit IgG (A10042; Life Technologies), and Alexa Fluor 568 donkey anti-goat IgG (A11057; Life Technologies).

Kretzschmar K., Post Y., Bannier-Hélaouët M., Mattiotti A., Drost J., Basak O., Li V.S., van den Born M., Gunst Q.D., Versteeg D., Kooijman L., van der Elst S., van Es J.H., van Rooij E., van den Hoff M.J, & Clevers H. (2018). Profiling proliferative cells and their progeny in damaged murine hearts. Proceedings of the National Academy of Sciences of the United States of America, 115(52), E12245-E12254.

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Characterization of Pancreatic Tissue

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Antibodies to GSK-3α/β, GSK-3β, phospho-GSK-3β (ser9), Ki-67, S6 ribosomal protein, phospho-S6 ribosomal protein (ser235, 236), phospho-S6 ribosomal protein (ser240, 244) were obtained from Cell Signaling Technologies (Beverly, MA), β-catenin was purchased from BD Transduction Lab (San Jose, CA), β-actin, α-amylase, and αSMA were from Sigma (St. Louis, MO), cytokeratin 19 was from DSHB (University of Iowa, Iowa). For immunohistochemical and immunofluorescence staining, the following primary antibodies were used: mouse anti-β-catenin (1:200), mouse anti-αSMA (1:200), rabbit anti-amylase (1:400), rat anti-CK19 (TROMAIII, 1:300), rabbit anti-GSK-3β (1:100), rabbit anti-phospho-S6 ribosomal protein (ser235, 236) at 1:300 dilution, and rabbit anti-Ki67 (1:200). For immunofluorescence staining, Alexa Fluor 488 donkey anti-mouse IgG, Alexa Fluor 568 donkey anti-rabbit IgG (Life Technologies), and Alexa Fluor 633 donkey anti-rat IgG (Jackson ImmunoResearch Laboratories) were used as secondary antibodies at a 1:300 dilution.

Ding L., Liou G.Y., Schmitt D.M., Storz P., Zhang J.S, & Billadeau D.D. (2017). Glycogen synthase kinase-3β ablation limits pancreatitis induced acinar-to-ductal metaplasia.. The Journal of pathology, 243(1), 65-77.

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