A31573

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The A31573 is a laboratory instrument designed for the detection and quantification of various analytes. It utilizes a specialized detection method to provide accurate and reliable measurements. The core function of this product is to facilitate precise analytical procedures in a laboratory setting.

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Close spelling variants of this product

Donkey anti-rabbit IgG Alexa647 (A31573, (2 citations) Alexa Fluor 647 (A31573 (2 citations)

88 protocols using a31573

Neddylation Pathway Transfection Assay

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Transfection was performed using Lipofectamine 2000 (Invitrogen, 11668-019) following the manufacturer’s protocol by combining reagent (µL) with DNA (µg) at a 2:1 ratio. DMSO was purchased from Sigma-Aldrich (D2650). MLN4924 was purchased from Active Biochemicals (A-1139).
Antibodies rabbit anti-CUL1 (ab75812), rabbit anti-CUL3 (ab194584), rabbit anti-NEDD8 (ab81264), and rabbit anti-CSN4 (ab12322) were purchased from Abcam. Donkey anti-rabbit secondary antibodies conjugated to Alexa Fluor 488, 555, or 647 were purchased from Invitrogen (A-21206, A-31572 and A-31573, respectively).

Chung D., Salsman J, & Dellaire G. (2019). Inhibition of neddylation induces mitotic defects and alters MKLP1 accumulation at the midbody during cytokinesis. Cell Cycle, 18(10), 1135-1153.

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Antibody Panel for Cytoskeletal Proteins

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The following antibodies were used: acetylated α-tubulin (T6793, clone 6-11B-1; Sigma) and βIII-tubulin (Tuj1, T8578, clone 2G10; Sigma), MBP (ab7349; Abcam), HA (11867431001, clone 3F10; Roche), contactin 1 (sc-20296; Santa Cruz Biotechnology). Rabbit polyclonals anti-Gal-4 and anti-Gal-3 (in-house production) were purified by affinity chromatography-based depletion, using the respective galectin as bead-immobilized ligand, and further tested by means of ELISA screening and Western blot against other commonly expressed members of this family, i.e. galectins-1, -2, -7, -8, and -9, to exclude cross-reactivity^{51 (link)}. F-actin was detected with TRITC- or FITC-conjugated phalloidin (P1951 and p5282, respectively, Sigma). Appropriate fluorescent anti-species secondary antibodies were from Invitrogen (A21202; A21203; A21206; A21207; A11055; A11058; A31571; A31573).

Díez-Revuelta N., Higuero A.M., Velasco S., Peñas-de-la-Iglesia M., Gabius H.J, & Abad-Rodríguez J. (2017). Neurons define non-myelinated axon segments by the regulation of galectin-4-containing axon membrane domains. Scientific Reports, 7, 12246.

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Immunofluorescence Protein Detection

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The primary protein antibodies to GAPDH (goat pAb; SAB2500450, Sigma), β-tubulin (rabbit pAb; ab6046, Abcam), α-Actinin (rabbit mAb, 6487S, Cell Signaling Technology), ER-α (rabbit mAb; RM9101S, Thermo Fisher Scientific), and mTOR (rabbit,2983S, Cell Signaling Technologies) were used. Secondary antibodies to goat IgG prelabelled with Alexa Fluor 647 (A21447), rabbit IgG pre-labelled with Alexa Fluor 555 or 647 (A31572 and A31573) were purchased from Invitrogen. All primary antibodies were used at a 1:10 dilution in 2% TBST/BSA from stock concentrations and incubated for 2 hours at room temperature, except for anti-GAPDH and anti-ER-α, which were incubated for 3 hours at room temperature. Secondary antibodies were diluted to a 1:20 working concentration in 2% TBST/BSA from stock and incubated for 1 hour at room temperature, protected from light.

Tan K.Y, & Herr A.E. (2020). Ferguson analysis of protein electromigration during single-cell electrophoresis in an open microfluidic device. The Analyst, 145(10), 3732-3741.

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Immunohistochemical Detection of Chlamydia and Amyloid-β

Cited 2 times

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Immunohistochemistry was performed as previously described^{27 (link),43 (link),44 (link)}. Specimens were incubated with goat anti-C. trachomatis/C. pneumoniae (this antibody is used to detect both of these Chlamydia species; Abcam ab20929; 1:400) and/or rabbit anti-Aβ peptide (Abcam ab201060,1:500). Secondary antibodies were donkey anti-goat Alexa Fluor 488 (Abcam ab150129 1:400), donkey anti-rabbit 647 (Invitrogen A31573; 1:500). Antibodies were diluted in blocking buffer (2% bovine serum albumin with 0.3% Triton X-100 in PBS). Cryostat sections were first incubated with blocking buffer for 60 min at room temperature, followed by overnight incubation with primary antibodies at 4 °C. Sections were washed for 3 × 5 min, then incubated with secondary antibodies for 1 h. Cell nuclei were stained with 4′6-diamidino-2-pheylindole (DAPI).

Chacko A., Delbaz A., Walkden H., Basu S., Armitage C.W., Eindorf T., Trim L.K., Miller E., West N.P., St John J.A., Beagley K.W, & Ekberg J.A. (2022). Chlamydia pneumoniae can infect the central nervous system via the olfactory and trigeminal nerves and contributes to Alzheimer’s disease risk. Scientific Reports, 12, 2759.

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Immunofluorescence Staining Antibody Protocol

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Primary antibodies used included those against α-tubulin (T5168, Sigma-Aldrich, ascites fluid, 1:5000; ab4074, Abcam, 1 μg ml⁻¹), β-tubulin (ab21057, Abcam, 1 µg ml⁻¹), spastin (ab31850, Abcam, 10 µg ml⁻¹; Fig. S1A-C), polyglutamylated tubulin B3 (T9822, Sigma-Aldrich, 2 µg ml⁻¹), γH2AX (05-636, Millipore, 0.1 µg ml⁻¹), cleaved caspase 3 (9664, Cell Signaling Technology, 0.5 μg ml⁻¹) and cleaved-caspase 9 (9509, Cell Signaling Technology, 1 μg ml⁻¹). Secondary antibodies included Alexa Fluor 488 donkey anti-goat (A11055, Invitrogen), Alexa Fluor 555 donkey anti-goat (A21432, Invitrogen), Alexa Fluor 555 donkey anti-mouse (A31570, Invitrogen), Alexa Fluor 647 donkey anti-mouse (A31571, Invitrogen) and Alexa Fluor 647 donkey anti-rabbit (A31573, Invitrogen). Parallel sections were processed in the absence of a primary antibody to control for secondary antibody specificity.

Cheers S.R., O'Connor A.E., Johnson T.K., Merriner D.J., O'Bryan M.K, & Dunleavy J.E. (2023). Spastin is an essential regulator of male meiosis, acrosome formation, manchette structure and nuclear integrity. Development (Cambridge, England), 150(6), dev201183.

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Microglial Phenotype Characterization by ICC

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We also analyzed the phenotype of microglia at day 4–5 after isolation. Previously, light microscopy images were taken before staining with antibodies using an AxioObserver Z1 (Carl Zeiss, Jena, Germany). For immunocytochemistry (ICC), the cells were fixed in 4% buffered formalin, washed with 0.1% Triton X-100 in PBS and stained with GFAP (ab16997, Abcam, Cambridge, UK), Iba1 (ab5076, Abcam), TNF-α (ab199013, Abcam) and TGF-β (ab92486, Abcam) antibodies at a working dilution of 1:100 for 1.5 h at RT, then washed and stained secondary antibodies. For staining the appropriate Iba1, Alexa Fluor 488 conjugated anti-goat secondary antibodies ab150129 (Abcam), and for staining the appropriate GFAP, TNF-α and TGF-β, Alexa Fluor 647 conjugated anti-rabbit secondary antibodies A-31573 (Invitrogen, Waltham, MA, USA) were used. After incubation for 30 min at RT, the cells were counterstained with 40,6-Diamidino-2-phenylindole (DAPI) (10 mg/mL in PBS, Sigma, St. Louis, MI, USA) to visualize the nuclei. The results were analyzed using an LSM 780 Confocal Microscope (Carl Zeiss, Jena, Germany).

Akhmetzyanova E.R., Zhuravleva M.N., Timofeeva A.V., Tazetdinova L.G., Garanina E.E., Rizvanov A.A, & Mukhamedshina Y.O. (2023). Severity- and Time-Dependent Activation of Microglia in Spinal Cord Injury. International Journal of Molecular Sciences, 24(9), 8294.

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Immunofluorescent Analysis of Tissue Samples

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Paraformaldehyde‐fixed (4%), paraffin‐embedded samples were stained with hematoxylin and eosin (H&E). For immunofluorescent analyses, samples were incubated for 1 h with CD31 antibody solution, dilution 1:200 (#ABIN 726140, antibodies‐online, Aachen, Germany), alpha smooth muscle actin (αSMA) antibody solution, dilution 1:200 (#ab7817, Abcam, Cambridge, United Kingdom), or Villin antibody solution, dilution 1:100 (#V1616C01, DCS, Hamburg, Germany), and anti‐pan cytokeratin (PCK) antibody solution, dilution 1:100 (C2562, Sigma‐Aldrich, Munich, Germany). For detection, Alexa Fluor^® 647 donkey anti‐mouse, dilution 1:400 (#A‐31571, Invitrogen, Pailey, United Kingdom), Alexa Fluor^® 647 donkey anti‐rabbit, dilution 1:400 (#A‐31573, Invitrogen, Pailey, United Kingdom), Alexa Fluor^® 555 donkey anti‐mouse, dilution 1:400 (#A‐31570, Invitrogen), and Alexa Fluor^® 555 donkey anti‐rabbit, dilution 1:400 (#A‐31572, Invitrogen) were used.

Schuerlein S., Schwarz T., Krziminski S., Gätzner S., Hoppensack A., Schwedhelm I., Schweinlin M., Walles H, & Hansmann J. (2016). A versatile modular bioreactor platform for Tissue Engineering. Biotechnology Journal, 12(2), 1600326.

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Immunostaining of Cardiomyocytes in Culture

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Isolated neonatal mouse cardiomyocytes or mouse HL-1 cells were cultured on μ-Slides and fixed with 4% formaldehyde (Thermo Fisher Scientific, 28908) for 10 minutes at room temperature. Subsequently, cells were permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature. Next, cells were blocked with 2% BSA (Sigma-Aldrich, A7030) and 1% donkey serum (Sigma-Aldrich, D9663) for 1 hour at room temperature, followed by primary antibody incubation at 4°C overnight in a humidified chamber. Cells were washed 3 times with PBS and incubated with secondary antibodies for 1 hour at room temperature. Phalloidin (Invitrogen, 1:200) was added together with the secondary antibodies to visualize the cytoskeleton. Immunostainings were imaged using a Zeiss LSM780 confocal microscope and analyzed using Volocity software 6.5.1.
The following primary and secondary antibodies were used: rabbit anti–troponin T (Abcam, ab45932; 1:1000), rabbit anti–Aurora B (Abcam, ab2254; 1:500), donkey anti-rabbit–Alexa Fluor 555 (Invitrogen, A-31572; 1:200), and donkey anti-rabbit–Alexa Fluor 555 (Invitrogen, A-31573; 1:200).

Aslan G.S., Jaé N., Manavski Y., Fouani Y., Shumliakivska M., Kettenhausen L., Kirchhof L., Günther S., Fischer A., Luxán G, & Dimmeler S. (2023). Malat1 deficiency prevents neonatal heart regeneration by inducing cardiomyocyte binucleation. JCI Insight, 8(5), e162124.

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Quantifying Neuronal Responses to AZ and AZ-Acid

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To analyze the number of neurons after AZ and AZ-acid treatment, cells were cultured and treated as described above, and on DIV 14, cells were fixed and then incubated with blocking buffer (PBS with 2% normal donkey serum and 0.5% Triton X-100) at room temperature for 30 min, followed by staining with neuronal nuclear antigen (NeuN; 1:1,000; ABN78, EMD Millipore) for 2 h. Cells were rinsed with PBS before applying AlexaFluor-conjugated donkey secondary antibody (1:1,000; A31573, Invitrogen) and DAPI (1:4,000). After a 1 h incubation with the secondary antibody, cells were washed three times with PBS and then observed by high-content microscopy as described above.

Hu W., Liu C.W., Jiménez J.A., McCoy E.S., Hsiao Y.C., Lin W., Engel S.M., Lu K, & Zylka M.J. (2022). Detection of Azoxystrobin Fungicide and Metabolite Azoxystrobin-Acid in Pregnant Women and Children, Estimation of Daily Intake, and Evaluation of Placental and Lactational Transfer in Mice. Environmental Health Perspectives, 130(2), 027013.

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Immunofluorescence Analysis of Engineered Stem Cells

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Immunofluorescence was performed according to previously reported methods (10 (link)). Transfected cMSCs (1.5×104 cells/cm2) were fixed with 4% paraformaldehyde (Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 15 min at room temperature, washed in phosphate-buffered saline (PBS; AR0030; Wuhan Boster Biological Technology, Ltd.), then treated with 0.2% Triton X-100 (T8787; Sigma-Aldrich) for 15 min. Cells were incubated with rabbit polyclonal anti-HCN4 antibody (1:100; ab69054; Abcam, Cambridge, UK) overnight at 4°C. Following washing three times with PBS for 5 min, the cells were incubated with Alexa FluorTM 647-conjugated donkey anti-rabbit IgG (1:200; A-31573; Invitrogen) for 60 min at 25±1°C. After further washing with PBS, the cells were mounted with Antifade Mounting Medium (Beyotime Institute of Biotechnology, Shanghai, China). Nuclei were stained with 4′,6-diamidino-2-phenylindole (D9542; Sigma-Aldrich) as a location control. Fluorescent images were obtained using an inverted laser confocal microscope (LSM 710; Carl Zeiss Microscopy GmbH, Cologne, Germany). The results were analyzed using ZEN lite software, 2011 edition (Carl Zeiss Microscopy GmbH).

FENG Y., LUO S., YANG P, & SONG Z. (2016). Electric pulse current stimulation increases electrophysiological properties of If current reconstructed in mHCN4-transfected canine mesenchymal stem cells. Experimental and Therapeutic Medicine, 11(4), 1323-1329.

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