The following primary antibodies were used: α-smooth muscle actin (αSMA), SM22α, CD31, CD34, von Willebrand factor (vWF), GFP, cardiac troponin T, anti-vascular endothelial growth factor receptor-2 (VEGFR-2), transforming growth factor β1 (TGF-β1), TGF-β receptor 1 (TGF-β R1), TGF-β receptor 2 (TGF-β R2), caspase-3, caspase-9, vinculin, mouse IgG polyclonal isotype control, rabbit IgG polyclonal isotype control, and goat IgG polyclonal isotype control (Abcam, Cambridge, MA). Fluorescent-conjugated secondary antibodies included: AlexaFluor®488 donkey anti-mouse IgG, AlexaFluor®594 donkey anti-rabbit IgG, AlexaFluor®594 donkey anti-mouse IgG, AlexaFluor®594 donkey anti-goat IgG, AlexaFluor®488 donkey anti-rabbit IgG, AlexaFluor®488 donkey anti-goat IgG, AlexaFluor®594 donkey anti-rabbit IgG, and AlexaFluor®647 donkey anti-mouse IgG (Abcam); horseradish peroxidase-conjugated secondary antibody (N-Histofine®, NICHIREI Biosciences Inc., Tokyo, Japan); 4′ 6-diamidino-2-phenylindole (DAPI, NucBlue® Fixed Cell ReadyProbes® Reagent, ThermoFisher Scientific, Waltham, MA).
Alexa fluor 594 donkey anti rabbit igg
Manufactured by Abcam
Sourced in United Kingdom, Japan
Alexa Fluor 594 donkey anti-rabbit IgG is a secondary antibody conjugated with the Alexa Fluor 594 dye. It is designed to detect and visualize rabbit primary antibodies in various immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 donkey anti rabbit igg, by Abcam (3 mentions)
Vibrating microtome, by Leica (2 mentions)
Glial fibrillary acidic protein (gfap), by Olympus (2 mentions)
Iba 1, by Abcam (2 mentions)
Alexa fluor 488 donkey anti mouse igg, by Abcam (2 mentions)
Ab24751, by Abcam (1 mentions)
Sab2108306, by Merck Group (1 mentions)
Nucblue fixed cell readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 donkey anti mouse igg, by Abcam (1 mentions)
Alexafluor 594 donkey anti goat igg, by Abcam (1 mentions)
Alexafluor 488 donkey anti goat igg, by Abcam (1 mentions)
Alexafluor 647 donkey anti mouse igg, by Abcam (1 mentions)
Mouse monoclonal anti neun, by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Abcam (1 mentions)
Rabbit polyclonal anti gfp, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 goat anti rat igg, by Abcam (1 mentions)
Ix81 zdc2, by Olympus (1 mentions)
Alexa fluor 594 goat anti mouse igg, by Abcam (1 mentions)
Protein ladder, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
6 protocols using alexa fluor 594 donkey anti rabbit igg
1
Comprehensive Immunohistochemical Analysis
2
Neuroinflammation and Cell Death Assessment
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Rats (21 d after reperfusion) were anesthetized with chloral hydrate and perfused transcardially with ice-cold saline followed by perfusion with 4% paraformaldehyde. The brains were extracted and dehydrated with a 10%, 20%, 30% sucrose gradient and then coronal sectioned (30 μm) using a vibrating microtome (Leica, Wetzlar, Germany). The sections were incubated in PBS (0.5% Triton X-100 and 10% goat serum) for 1 h at room temperature, following by incubation with rabbit polyclonal, TUNEL (Elabscience, CHN), GFAP (CST, USA), or rabbit polyclonal Iba-1 (Abcam, UK) at 4 °C overnight. After several PBS rinses, sections were incubated with Alexa Fluor 594 donkey anti-rabbit IgG (Abcam, UK) or Alexa Fluor 488 donkey anti-rabbit IgG (Abcam, UK). The number of TUNEL, GFAP or Iba1-positive cells was analyzed by fluorescence confocal microscopy (EX61, Olympus, Tokyo, Japan). The positive cells were counted in three randomly chosen squares of identical size (460 × 460 μm).
3
Immunofluorescence Imaging of SOX4 and p180
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Following drying in a 60°C oven overnight, the tissue sections were deparaffinized in xylene and rehydrated in a gradient ethanol series. For antigen retrieval, the sections were microwaved in 10 mM citrate buffer (pH 6.0) for 20 min. The sections were then incubated in 5% bovine serum for 1 h at 37°C. A combination of two primary antibodies, rabbit polyclonal anti-SOX4 (SAB2108306; 1:100 dilution; Sigma-Aldrich Merck KGaA, St. Louis, MO, USA) and mouse monoclonal anti-p180 (ab24751; 1:50 dilution; Abcam, Cambridge, UK), was used for incubating the sections overnight at 4°C, while for negative controls PBS was used in the place of the antibody. The sections were then incubated with a mixture of two secondary antibodies, Alexa Fluor-594 donkey anti-rabbit IgG (ab150076; 1:500 dilution; Abcam) and Alexa Fluor-488 donkey anti-mouse IgG (ab150105; 1:500 dilution; Abcam) for 4 h at room temperature, followed by DAPI incubation for nuclear staining. Double immuofluoresence images were acquired using a confocal laser-scanning microscope (C1; Nikon, Tokyo, Japan).
4
Rat Brain Histological Analysis
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Rats (21 d after reperfusion) were anesthetized with chloral hydrate and perfused transcardially with ice-cold saline and subsequently paraformaldehyde (4%). The brains were extracted and dehydrated with gradient sucrose (10, 20, and 30%) and then coronal sectioned (30 μm) with a vibrating microtome (Leica, Wetzlar, Germany). The sections were incubated in PBS (0.5% Triton X-100 and 10% goat serum) for 60 min at room temperature, following by incubation with rabbit polyclonal, TUNEL (Elabscience, CHN), rabbit polyclonal Iba-1 (Abcam, Cambridge, UK), or GFAP (CST) at 4 °C overnight. After three PBS rinses, sections were incubated with Alexa Fluor 594 donkey anti-rabbit IgG (Abcam, Cambridge, UK), or Alexa Fluor 488 donkey anti-rabbit IgG (Abcam, Cambridge, UK). The number of TUNEL, GFAP, or Iba1 positive cells was observed by fluorescence confocal microscopy (EX61, Olympus, Tokyo, Japan). The positive cells were counted in three randomly chosen squares of identical size (460 × 460 μm).
5
Immunohistochemical Analysis of Cell Markers
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Immunohistochemical staining was performed as previously reported (Zhang et al., 2015). On day 7 or 21 following PCI, the brains were removed, and 5-µm frozen coronal sections were prepared. After antigen retrieval, sections were immunolabeled with rat monoclonal anti-BrdU (1:500; Accurate Chemical & Scientific), rabbit polyclonal anti-GFP (1:200; Santa Cruz Biotechnology), mouse monoclonal anti-NeuN (1:500; Chemicon), rabbit monoclonal anti-GFAP (1:200; Chemicon), mouse monoclonal anti-RIP (1:50; kind gift from Dr Xu XM, University of Louisville School of Medicine) and mouse monoclonal anti-NgR1 (1:200; Biogen Idec, Inc.) overnight at 4°C. After washing with PBS, the sections were incubated with goat anti-rabbit IgG Alexa Fluor 488, goat anti-rat IgG Alexa Fluor 488, goat anti-mouse IgG Alexa Fluor 594 and donkey anti-rabbit IgG Alexa Fluor 594 (1:1000; Abcam) at room temperature for 1 hour. Cells double-positive for BrdU/NeuN, GFP/GFAP and GFP/RIP were counted. Positive cells were also visualized using a motorized inverted microscope (IX81-ZDC2; Olympus, Hamburg, Germany).
6
Echinacoside Modulates STAT3 Signaling
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Echinacoside was purchased from Selleck and dissolved into 5, 10, and 20 mg/L. LPS were from Sigma. STAT3 inhibitor, S3I-201, was purchased from Selleck. Opti-MEM was from Gibco. Antibodies used in this study such as those against tyrosine hydroxylase (TH), against α-synuclein, and against brain-derived neurotrophic factor (BDNF) were purchased from Cell Signaling Technology (CST, United States); that against ionized calcium-binding adapter molecule 1 (IBA-1, goat) was from Novus Bio; those against STAT3, p-STAT3(tyr705), p-STAT3(ser727), donkey anti-rabbit IgG (Alexa Fluor® 594), and donkey anti-goat IgG (Alexa Fluor® 488) were from Abcam (United States); and those against IL-6, p-JAK2 (tyr1007/1008), JAK2, and β-actin were from ABclonal (China). SDS-PAGE gel preparation and cocktail protease inhibitor were purchased from Servicebio (China). Universal antibody diluent and serum-free cell freezing medium were purchased from New Cell and Molecular Biotech (NCM, China). Protein Ladder and SuperSignal West Pico PLUS Chemiluminescent Substrate were purchased from Thermo (United States).
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