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Goat anti rabbit igg conjugated with alexa fluor 594

Manufactured by Thermo Fisher Scientific

Goat-anti-rabbit IgG conjugated with Alexa Fluor 594 is a secondary antibody reagent used in immunodetection techniques. It consists of goat-derived antibodies that specifically bind to rabbit primary antibodies, and are conjugated with the Alexa Fluor 594 fluorescent dye. This product can be used to detect and visualize target proteins in various applications, such as Western blotting, immunohistochemistry, and flow cytometry.

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6 protocols using goat anti rabbit igg conjugated with alexa fluor 594

1

Tracking Stem Cell Migration and Neuronal Response in MCAO

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Tissues of MCAO+ SKP-SCs group were directly observed under a fluorescence microscope (ZEISS, Germany) to detect survival and migration of GFP-labeled SKP-SCs in the rat brain at 6 h, 12 h on Day 0, Days 1, 4, 7, 14 and 28 after MCAO. To further observe the health of neurons and glial cells, as well as neuronal dendritic processes and axonal growth, additional sets of slides were stained with antibodies against different neuronal and glial cell markers, including NeuN, GFAP, MAP2, and GAP43. After temperature equilibration and drying, sections mounted on slides were washed with PBS for 10 min, then blocked with 0.1% Triton x-100 for 2 h, before being incubated overnight at 4°C with the following antibodies: rabbit anti-NeuN antibody (1:500, Abcam, USA) and rabbit anti-GFAP antibody (1:500, Abcam, USA), mouse anti-MAP2 antibody (1:500, Abcam, USA), and rabbit anti-GAP43 antibody. Indirect fluorescence by incubating sections at room temperature for 2 h with goat-anti-rabbit IgG conjugated with Alexa Fluor 594 or goat-anti-mouse-IgG conjugated with Alexa Fluor 488 (1:1000, Life Technologies) was used for detection. Sections were further incubated with Hoechst for 10 min before being coverslipped for microscopic observation.
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2

Immunofluorescence Assay for EV-D68 Infection

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SK-N-SH and RD cells (∼80% confluent) in a 96-well plate were infected with EV-D68 at an MOI of 2. Mock-infected cells were included as a negative control. EV-D68-infected SK-N-SH and RD cells were incubated at 37°C and 33°C in 5% CO2, respectively. After 8 hpi, cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, washed with PBS, and permeabilized with 70% ethanol. Cells were first incubated with 5% bovine serum albumin (BSA; Aurion, Wageningen, The Netherlands) in PBS for 30 min before incubation with rabbit anti-EV-D68 VP1 (20 μg/ml; GeneTex, Irvine, CA, USA) for 1 h. Cells were washed twice with PBS and incubated with goat anti-rabbit IgG conjugated with Alexa Fluor 594 (10 μg/ml; Life Technologies, Inc., The Netherlands) in PBS with 0.1% BSA (Aurion) for 1 h. Cells were washed 3 times with PBS and mounted with ProLong Diamond Antifade with DAPI (4′,6-diamidino-2-phenylindole; Life Technologies) to visualize nuclei. Each experiment included negative and omission controls. EV-D68 VP1-positive cells were identified using a Zeiss LSM 700 laser scanning microscope. All images were processed using Zen 2010 software. Per sample, 3 high-power fields were photographed, and the number of infected cells was calculated by counting virus-infected/uninfected cells in 3 randomly chosen panels. All experiments were performed in triplicate.
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3

Fluorescently Labeled Antibody Probes

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Goat anti‐guinea pig (Cat# A11073) or mouse IgG conjugated with Alexa Fluor 488 (Cat# A11029) and goat anti‐rabbit IgG conjugated with Alexa Fluor 594 (Cat# A11037) were purchased from ThermoFisher Sci, Waltham, MA.
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4

Immunofluorescence Analysis of Ocular CD4+, IL-17+, and IFN-γ+ Cells

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The left eyeballs were embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, catalog no. 4583; Sakura Finetek USA, Torrance, CA, USA). Frozen sections (5 µm thick) were fixed with acetone for 15 minutes at –20°C. Subsequently, the samples were washed three times with PBS, followed by blocking with 2% BSA in PBS for 1 hour at room temperature. The samples were then incubated with monoclonal rabbit anti-CD4 antibody (1:200, 3.475 µg/mL, catalog no. ab183685; Abcam, Inc., Cambridge, MA, USA) and counterstained with polyclonal rabbit anti-IL-17 antibody (1:200, 5 µg/mL, catalog no. ab79056; Abcam) and monoclonal mouse anti-IFN-γ antibody (1:50, 4 µg/mL, catalog no. sc-12755; Santa Cruz Biotechnology, Dallas, TX, USA) diluted with 1% BSA overnight at 4°C. After they were washed three times with PBS, the samples were incubated in secondary antibody (goat-anti-rabbit IgG conjugated with Alexa Fluor 488, goat-anti-rabbit IgG conjugated with Alexa Fluor 594, or goat-anti-rat IgG conjugated with Alexa Fluor 594; Thermo Fisher Scientific) diluted with 1% BSA for 1 hour at room temperature. Finally samples were counterstained with 4′,6-diamidino-2-phenylendole (DAPI, catalog no. H-1200; Vector Laboratories, Inc., Burlingame, CA, USA). The results were photographed with a Leica upright microscope (DM2500; Leica Microsystems, Wetzlar, Germany).
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5

Immunocytochemical Analysis of iPSC-Derived Neurons

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Pre-differentiated iPSC-NPs plated on poly-L-ornithine and laminin-coated chamber slides were washed in PBS and fixed with 4% paraformaldehyde in PBS for 15 min. Fixed cells were washed twice with PBS before staining. To identify neural precursors and differentiated neurons, antibodies directed against βIII-tubulin (1:100), Olig2 (1:400), nestin (1:200), chondroitin sulfate proteoglycan (NG2, 1:400), NF70 (1:200) (all Sigma-Aldrich), and Ki-67 (1:50) (Abcam) were used. To visualize primary antibodies, goat anti-mouse IgG conjugated with Alexa-Fluor 488 (1:400) and goat anti-rabbit IgG conjugated with Alexa-Fluor 594 (1:400) (Molecular Probes, Eugene, OR) were used. To visualize cell nuclei, DAPI was used. Confocal images were taken with the Zeiss LSM 5 Duo confocal microscope (Carl Zeiss AG). The index of the mitotic activity of the cells was evaluated as the ratio of Ki-67 positive cells to the total number of cells in the 10 randomly selected fields of view.
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6

Immunofluorescent Profiling of Malaria Parasites

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Blood smears of asynchronous 3D7 strain parasite cultures were prepared, fixed in cold methanol for 15 min and probed with anti-PfGBP130-A generated by DNA, mRNA or recombinant proteinbased immunization (dilutions tested from 1:50 to 1:200), and rabbit anti-PfMSP4 (obtained from MR4) diluted 1:500 in PBS, 5% BSA, pH 7.4. Blood smears were incubated with primary antibodies for 1 h at 25°C, washed three times in PBS, 0.05% Tween 20, and incubated with goat anti-mouse IgG conjugated with Alexa Fluor 488 (Molecular Probes) and goat anti-rabbit IgG conjugated with Alexa Fluor 594 (Molecular Probes). Blood smears were incubated for 10 min in 1 mg/ml of DAPI (Sigma-Aldrich) to label nuclei and cover slipped with ProLong Gold antifade reagent (Invitrogen). Blood smears were imaged using a confocal microscope (ZEISS LSM 900 Airyscan) equipped with a ×63 oil-immersion objective.
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