Alexa fluor 594 goat anti rabbit igg antibody

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Alexa Fluor® 594 goat anti-rabbit IgG antibody is a secondary antibody conjugated with the Alexa Fluor® 594 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassay and imaging applications.

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Alexa Fluor 594 goat anti-rabbit IgG secondary antibody Alexa Fluor 594–conjugated goat polyclonal anti-rabbit IgG secondary antibody Alexa Fluor® 594 goat anti-rabbit IgG (H+L) antibody Alexa Fluor 594-labeled goat anti-rabbit IgG antibodies Alexa Fluor 594 goat anti-rabbit IgG (H+L) secondary antibody Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody Alexa Fluor 594-conjugated Goat anti-Rabbit IgG (H+L) secondary Antibody Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody Alexa Fluor™ 594 Goat anti-rabbit IgG cross-absorbed secondary antibody conjugated to Alexa Fluor 594 Alexa Fluor 594-conjugated goat anti-rabbit IgG secondary antibody

20 protocols using alexa fluor 594 goat anti rabbit igg antibody

Immunofluorescence Analysis of TGF-β Signaling

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TRIzol Reagent, AlexaFluor 594 goat anti-rabbit IgG antibody, AlexaFluor 594 donkey anti-rabbit IgG antibody, AlexaFluor 488 donkey anti-goat IgG antibody and Hoechst 33258 nuclear stain were purchased from Life Technologies (Grand Island, NY). Rabbit anti-aquaporin-5 polyclonal antibody (178615) was purchased from EMD Millipore (Billerica, MA). Rat anti-CD45 monoclonal antibody (30-F11) was purchased from Biolegend (San Diego, CA). Rabbit anti-TGF-β1/2/3 polyclonal antibody (3771), rabbit anti-Smad2/3 monoclonal antibody (D7G7), rabbit anti-phospho-Smad2/3 monoclonal antibody (D27F4) and rabbit anti-E-cadherin monoclonal antibody (24E10) were purchased from Cell Signaling Technology (Danvers, MA). Rabbit anti-Snail polyclonal antibody (NBP1-19529) was purchased from Novus Biologicals (Littleton, CO). Rabbit anti-TGF-β R1 polyclonal antibody (H-100), goat anti-aquaporin-5 polyclonal antibody (G-19) and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The TGF-β R1 inhibitors SB431542 and GW788388 were purchased from Tocris Bioscience (Bristol, United Kingdom). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless stated otherwise.

Woods L.T., Camden J.M., El-Sayed F.G., Khalafalla M.G., Petris M.J., Erb L, & Weisman G.A. (2015). Increased Expression of TGF-β Signaling Components in a Mouse Model of Fibrosis Induced by Submandibular Gland Duct Ligation. PLoS ONE, 10(5), e0123641.

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Immunofluorescent Staining of Ileal Tight Junctions

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Frozen sections of ileal tissue were fixed with 1% paraformaldehyde in PBS containing 1 mmol/L CaCl₂ for 30 min at room temperature, then washed thrice with PBS for 5 min, permeabilized in 1% Triton X-100 in PBS at room temperature for 5 min. After washing with PBS, the non-specific binding sites were blocked with 5% normal goat serum in PBS for 30 min. Then, the sections were incubated with monoclonal rabbit antibody against ZO-1, occludin, claudin-1 or claudin-2 (Invitrogen) diluted at 1:200 with 1% normal goat serum in PBS at 4°C overnight. After washing thrice in PBS, sections were incubated with secondary Alexa Fluor 594 goat anti-rabbit IgG antibody (Life Technologies) at 1:200, Alexa Fluor 488-conjugated phalloidin (Invitrogen) at 1:100, and DAPI (Sigma) at 1:1,000 for 1 h at room temperature. After washing thrice in PBS, sections were mounted using Slowfade reagents (Invitrogen). Images were obtained using a TCS SP5 laser confocal microscopy (Leica, Germany).

Huang Y., Feng Y., Wang Y., Wang P., Wang F, & Ren H. (2018). Severe Burn-Induced Intestinal Epithelial Barrier Dysfunction Is Associated With Endoplasmic Reticulum Stress and Autophagy in Mice. Frontiers in Physiology, 9, 441.

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Immunofluorescence Analysis of ASC Cytokine Expression

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To analyze the ASC cytokine expression, ASCs were cultured on the coverslips (VWR International) until confluence. The ASCs were then fixed with 4% paraformaldehyde (Alfa Aesar, Ward Hill, MA, USA) in PBS for 1 h, and treated with 0.1% Triton X-100 (Sigma-Aldrich) for 15 min. After blocking with 3% bovine serum albumin (BSA, Sigma-Aldrich) in PBS at room temperature for 1 h, the slides were incubated with rabbit polyclonal anti-vascular endothelial growth factor (VEGF) antibody (1:100 dilution, sc-507; Santa Cruz Biotechnology), goat polyclonal anti-insulin-like growth factor-1 (IGF-1) antibody (1:100 dilution, sc-1422; Santa Cruz Biotechnology), or rabbit polyclonal anti-hepatocyte growth factor (HGF) antibody (1:100 dilution, sc-7949; Santa Cruz Biotechnology) at 4°C overnight. After incubation with secondary antibody Alexa Fluor 594 goat anti-rabbit IgG antibody (1:200 dilution, A11012; Life Technologies) or Alexa Fluor 594 donkey anti-goat IgG antibody (1:200 dilution, A11058; Life Technologies) at room temperature for 1 h, cell nuclei were counterstained with 4¢,6-diamidino-2-phenylindole dihydrochloride (DAPI; Santa Cruz Biotechnology).
The images were taken with a fluorescence microscope (Zesis Axio observer Z1 Inverted microscope; Carl Zesis Canada Ltd., Toronto, ON, Canada).

Chi C., Wang F., Xiang B., Deng J., Liu S., Lin H.Y., Natarajan K., Li G., Wang L., Wang J., Lin F., Freed D.H., Arora R.C., Liu H, & Tian G. (2015). Adipose-derived stem cells from both visceral and subcutaneous fat deposits significantly improve contractile function of infarcted rat hearts. Cell transplantation, 24(11).

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Subcellular Localization of CDCA7

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KYSE150, KYSE180, and KYSE450 cells were transfected with CDCA7-V5 plasmid and empty vector, respectively. Formaldehyde (4%) was used to fix the cells for 10 min. BSA (1%) was used to incubated the cells for 1 h to block non-specific protein–protein interactions after permeabilized by 0.1% Triton X-100. The cells were incubated with the primary antibody rabbit anti-V5 (Abcam, Cambridge, UK, 2 µg/ml) overnight at 4°C. Alexa Fluor^® 594 goat anti-rabbit IgG antibody (Thermo Fisher, Carlsbad, USA, 1:1,000) was used for 30 min at room temperature after washing four times in PBS. DAPI at a concentration of 0.5 µg/ml was used to stain the cell nuclei.

Li H., Weng Y., Wang S., Wang F., Wang Y., Kong P., Zhang L., Cheng C., Cui H., Xu E., Wei S., Guo D., Chen F., Bi Y., Meng Y., Cheng X, & Cui Y. (2021). CDCA7 Facilitates Tumor Progression by Directly Regulating CCNA2 Expression in Esophageal Squamous Cell Carcinoma. Frontiers in Oncology, 11, 734655.

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Immunofluorescence Analysis of HA-ZNF750

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HA-ZNF750wt plasmid and empty vector were transfected into KYSE140 cells respectively. Then the cells were 4% formaldehyde fixed for 10 min, permeabilized with 0.1% Triton X-100 for 5 min and then incubated in 1%BSA for 1 hr to block non-specific protein-protein interactions. The cells were incubated with rabbit anti-HA antibody (Abcam, Cambridge, UK, 5 µg/ml) overnight at 4 °C, followed by Alexa Fluor® 594 goat anti-rabbit IgG antibody (Thermofisher, Carlsbad, USA, 1:1000) for 30 min and washed three times in PBS. DAPI was used to stain cell nuclei at a concentration of 0.5 µg/ml. Images were acquired by the Olympus IX71 imaging system (×200).

Kong P., Xu E., Bi Y., Xu X., Liu X., Song B., Zhang L., Cheng C., Yan T., Qian Y., Yang J., Ma Y., Cui H., Zhai Y., Zou B., Liu X., Cheng Y., Guo S., Cheng X, & Cui Y. (2020). Novel ESCC-related gene ZNF750 as potential Prognostic biomarker and inhibits Epithelial-Mesenchymal Transition through directly depressing SNAI1 promoter in ESCC. Theranostics, 10(4), 1798-1813.

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Immunofluorescence Assay for E-cadherin

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Cells were seeded on slides in 12‐well plates. After culture for 12–24 h, the cells were fixed in 4% formaldehyde for 30 min at room temperature. Then the cells were washed with PBS and permeabilized with 0.25 Triton X‐100 for 30 min. The slides were blocked with 0.5% BSA in PBS for 1 h at room temperature after being washed with PBS four times. The cells were incubated with rabbit antibody against E‐cadherin (Proteintech, Cat no. 20874‐1‐AP, 1:100) overnight at 4°C. Alexa Fluor 594 goat anti‐rabbit IgG antibody (Thermo Fisher) was used for 1 h at room temperature. Then the cells were washed with PBS four times and stained with 0.5 μg/mL DAPI, followed by imaging using a confocal microscope (Leica TCS SP8).

Li H., Wang S., Li X., Weng Y., Guo D., Kong P., Cheng C., Wang Y., Zhang L., Cheng X, & Cui Y. (2022). CDCA7 promotes TGF‐β‐induced epithelial–mesenchymal transition via transcriptionally regulating Smad4/Smad7 in ESCC. Cancer Science, 114(1), 91-104.

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Quantifying Autophagy in THP-1 Cells

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THP-1 cells were seeded in 6-well plates with microscope coverslips. After the indicated treatments, cells were fixed with 10% paraformaldehyde and permeabilized in PBS containing 0.5% Triton X-100 (Sigma-Aldrich). The cells were incubated at 4°C overnight with rabbit anti-LC3 (Cell Signaling Technology, #12741). The secondary antibody was Alexa Fluor 594 goat anti-rabbit IgG antibody (Thermo Fisher Scientific, A-11012). Images were acquired using fluorescence microscopy. The percentage of autophagic cells with LC3 puncta (>10) was determined by counting ≥100 cells from three independent experiments. The size and number of LC3 puncta/cell were calculated using the ImageJ program.

Wang X., Ribeiro M., Iracheta-Vellve A., Lowe P., Ambade A., Satishchandran A., Bukong T., Catalano D., Kodys K, & Szabo G. (2019). Macrophage-specific HIF-1α contributes to impaired autophagic flux in non-alcoholic steatohepatitis. Hepatology (Baltimore, Md.), 69(2), 545-563.

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Oxidative Stress Response in Trypanosoma

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Wild type bloodstream cultures were grown in HMI-11 medium for 24 h up to a density of 5 × 10⁵ parasites/ml, and treated with 250 µM of H₂O₂ for 10 minutes in the same culture media. Transgenic procyclic cultures were grown in SDM-79 medium up to a density of 5 × 10⁶ parasites/ml and treated with 500 µM of H₂O₂ for 10 minutes.
Parasites were fixed with 3.8% (W/V) formaldehyde in PBS at 4 °C, permeabilized with fresh PBS-0.1% Triton X-100 and blocked at room temperature for 1 h. PAR was detected with 1:500 rabbit polyclonal anti- PAR antibody (BD) followed by 1:500 Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen). TbPARP was detected with 1:100 rabbit polyclonal anti-TbPARP antibody (GenScript), followed by 1:500 Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen). Fusion proteins TBP-PARP-eYFP, TBP-N-eYFP and TBP-WRA-eYFP were localized with fluorescence of eYFP. Excess of antibody was removed by 3 × 5 min washes in PBS, and nuclear and kinetoplast DNA stained with 2 μg/ml DAPI (Sigma). Coverslips were washed with distilled water and mounted in Mowiol and then visualized using an Olympus BX41 microscope.

Haikarainen T., Schlesinger M., Obaji E., Fernández Villamil S.H, & Lehtiö L. (2017). Structural and Biochemical Characterization of Poly-ADP-ribose Polymerase from Trypanosoma brucei. Scientific Reports, 7, 3642.

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Microscopic Quantification of Toxoplasma Replication

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For the data in Fig. 5A, infected HFF cells were fixed with 4% paraformaldehyde 18 and 27 h postinfection in chamber slides. Infected monolayers were permeabilized with 0.1% Triton X-100 and stained with rabbit anti-SAG1 antibody (1:1,000) followed by incubation with Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen) (1:1,000), mounting in Mowiol, and visualization by fluorescence microscopy. At least 100 vacuoles were counted from at least 6 fields of view for each experiment. For the data in Fig. 5B, chamber slides of infected HFF cells were fixed and stained (0.2% crystal violet in 70% ethanol for 7 min) at the indicated time points, washed three times for 7 min each in PBS, and mounted in Mowiol for light microscopy. At least 50 vacuoles were counted from at least 6 fields of view for each experiment. For bioluminescence analysis of replication, individual wells of HFF cells in a 96-well plate were inoculated with 1,000 parasites for 4 h before noninvaded parasites were washed away. The end of the invasion period was designated 0 h. At 0, 20, 32, 44, 56, and 68 h, the medium was removed, and infected cells were lysed with d-luciferin solution. Bioluminescence intensities were acquired by Bio-Tek Synergy HT microplate reader. Readings were normalized to the 0-h value set to 1 to account for interexperiment differences in initial parasite infectivity.

Dou Z., McGovern O.L., Di Cristina M, & Carruthers V.B. (2014). Toxoplasma gondii Ingests and Digests Host Cytosolic Proteins. mBio, 5(4), e01188-14.

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Immunofluorescence Staining of Prostate Tissue

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The tumor samples from prostate tumors in situ were fixed in 4% neutral buffered para-formaldehyde and embedded in paraffin. Prostate sections were deparaffinized in xylene solution and rehydrated using gradient ethanol concentrations. Sections were antigen retrieved and washed, blocked in PBS containing 5% BSA for 1 hr at room temperature in a humidified chamber. Sections were washed again and incubated with Pref-1 antibody (mouse) and CD34 (Rabbit) overnight at 4°C. Sections were washed with PBS and incubated with Invitrogen Alexa Fluor 488 goat anti-mouse IgG antibody (1:500) and Alexa Fluor 594 goat anti-rabbit IgG antibody (1:500) for 1 hr in the dark at room temperature. Slides were washed with PBS, stained with DAPI for 5 min, added with Thermal mountant permafluor and sealed with cover glasses. Sections were observed under fluorescence microscope and images were captured.

Xie H., Li L., Zhu G., Dang Q., Ma Z., He D., Chang L., Song W., Chang H.C., Krolewski J.J., Nastiuk K.L., Yeh S, & Chang C. (2015). Infiltrated pre-adipocytes increase prostate cancer metastasis via modulation of the miR-301a/androgen receptor (AR)/TGF-β1/Smad/MMP9 signals. Oncotarget, 6(14), 12326-12339.

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