Formalin-fixed, paraffin-embedded monkey TM sections were used for immunochemistry staining. After deparaffinizing and antigen retrieval, two antibodies were applied to the same specimen. A mouse monoclonal antibody of 8-OHdG, (sc-393871; Santa Cruz Biotechnology, Dallas, TX, USA) was used at 1:500 dilution, and a rabbit polyclonal antibody of fibronectin (ab23751; Abcam, Cambridge, MA, USA) was used at 1:500 dilution at 4°C overnight, respectively. Alexa-Fluor 488 donkey anti-mouse IgG and Alexa-Fluor 555 donkey anti-rabbit IgG (1/1000; Abcam) were used as secondary antibodies to detect 8-OHdG and fibronectin separately. 4′,6-Diamidino-2-Phenylindole (DAPI) was used for nuclear staining. ImageJ (http://imagej.nih.gov/ij/ ; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) was used for fluorescence intensity analysis.
Alexa fluor 488 donkey anti mouse igg
Manufactured by Abcam
Sourced in United States
Alexa Fluor 488 donkey anti-mouse IgG is a secondary antibody conjugated with the Alexa Fluor 488 fluorophore. It is designed to detect and label mouse immunoglobulin G (IgG) in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 555 donkey anti rabbit igg, by Abcam (3 mentions)
Alexa fluor 594 donkey anti rabbit igg, by Abcam (2 mentions)
Alexa fluor 555 donkey antigoat igg, by Abcam (2 mentions)
Ab23751, by Abcam (1 mentions)
Sc 393871, by Santa Cruz Biotechnology (1 mentions)
Ab24751, by Abcam (1 mentions)
Sab2108306, by Merck Group (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Abcam (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)
Ab150105, by Abcam (1 mentions)
T6793, by Merck Group (1 mentions)
Ab150074, by Abcam (1 mentions)
Tf0808, by Matsunami (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Fujifilm (1 mentions)
Fluoro keeper antifade reagent, by Nacalai Tesque (1 mentions)
Orca r2, by Hamamatsu Photonics (1 mentions)
8 protocols using alexa fluor 488 donkey anti mouse igg
1
Immunofluorescent Analysis of 8-OHdG and Fibronectin
2
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).
3
Comprehensive Immunohistochemical Analysis
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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).
4
Immunostaining of Sperm Axonemal Proteins
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Spermatozoa in Hank’s buffer were attached to the wells of an eight-well glass slide (TF0808; Matsunami). After washing out excess sperm, attached spermatozoa were briefly demembranated using 1% Nonidet P-40 for 2 min. Specimens were fixed with 2% paraformaldehyde/Hank’s buffer for 10 min at room temperature, followed by treatment with cold acetone and methanol (−20°C). After rehydration with PBST (phosphate buffered saline containing 0.1% Tween-20), specimens were treated with blocking buffer (2% normal goat serum, 1% cold fish gelatin in PBST). Immunostaining was performed with anti-acetylated tubulin antibody (1:500 dilution; T6793; Sigma-Aldrich) and anti-Dnah8 antibody (1:50 dilution; generated in this study) as primary antibodies. Alexa Fluor 488 Donkey anti-mouse IgG (1:250 dilution; ab150105; Abcam) and Alexa Fluor 555 Donkey anti-rabbit IgG (1:250 dilution; ab150074; Abcam) were used as secondary antibodies with 2.5 μg/ml DAPI (Wako) for nuclear staining. Specimens were mounted with Fluoro-KEEPER Antifade Reagent (Nacalai tesque) and observed with a fluorescence microscope (BX60; Olympus) and a CCD camera (ORCA-R2; Hamamatsu).
5
Immunofluorescence Staining of CD31 in HUVECs
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The co-cultured HUVECs for CD31 antibody as an endothelial cells marker were fixed with 4% paraformaldehyde (PFA (Sigma-Aldrich)) for 30 min at room temperature. The cells were permeabilized with 0.2% Triton-X 100 (Sigma-Aldrich) for 10 min, the cells were blocked with goat serum and incubated overnight at 4 °C with primary antibody CD31, mouse monoclonal anti-human (Abcam, 1:200), then to determine the specificity of immunofluorescence, labeled secondary antibody (Alexa Fluor@488 donkey anti mouse IgG, at a 1:500 dilution; Abcam, USA) was added for 2 h at room temperature in the dark. For negative controls, only the secondary antibody was used. After PBS washes, the nuclei of cells were stained by 4’,6-diamidino-2- phenylindole (DAPI, Sigma) in darkness for 5 min. The images were captured by fluorescence microscopy (Olympus BX51, Japan).
6
Immunofluorescence Staining of Bioartificial Pancreas
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To perform immunofluorescence staining of retrieved bioartificial pancreas, the histological sections were incubated with primary antibodies (mouse anti-insulin, Boster, China) and fluorescent secondary antibodies (Alexa Fluor 488 donkey anti-mouse IgG, Abcam, England) successively. The sections were further stained with DAPI staining (Beyotime, China) before observation under an inverted fluorescence microscope.
7
Quantifying Motor and Sensory Innervation in Fascia
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Tissue block specimens used for micro-CT analysis then underwent routine histological processing and cut into 5-µm-thick sections, which were stained using Masson’s trichrome and Verhoeff-Van Gieson to compare the fascial components of the proximal and distal parts of the AC. Choline acetyltransferase (ChAT) selectively labels peripheral motor axons in humans [13 (link)]. To quantify motor and sensory makeup of the pmNVM and SN, sections were incubated with the primary antibody against neurofilament 200 (NF200; mouse, diluted 1:400; Abcam) and ChAT (goat, diluted 1:100; Abcam). Antigens were observed in each section using the Abcam Alexa Fluor 488 donkey antimouse IgG and Abcam Alexa Fluor 555 donkey antigoat IgG. The ratio of ChAT-positive neurons to the cross-sectional area of each fascicle was computed in microns squared for each cross-sectional image (Image J version 1.53n, NIH, USA).
8
Quantifying TFEB Nuclear Translocation
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Antibodies were prepared in the following concentrations: TFEB 1:100 (Abcam), p62 1:100 (BD Biosciences, San Jose, CA, United States) and LAMP-1 1:100 (Santa Cruz Biotechnology, Dallas, TX, United States); secondary antibodies were Alexa Fluor 555 donkey anti-goat IgG 1:100 (Abcam), Alexa Fluor 647 donkey anti-goat IgG 1:100 (Thermo Fisher Scientific), Alexa Fluor 555 donkey anti-rabbit IgG (Abcam) and Alexa Fluor 488 donkey anti-mouse IgG 1:100 (Abcam). DAPI (Cell Signaling Technology) was used at a concentration of 1:10,000. Slides were viewed on a Zeiss LSM 700 confocal microscope (Carl Zeiss Canada, Toronto, ON, Canada). TFEB nuclear translocation was assessed on Adobe Photoshop (CS4) (San Jose, CA, United States) as the number of red pixels in at least three randomly selected x630 fields, in at least three experimental replicates per condition for NRK-52E cells or in kidney sections from vehicle- (n = 5) or Tubastatin A- (n = 4) treated rats.
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