Cells (5 × 105) were seeded in 6-cm dishes and incubated for 18 h at 37 °C in a humidified incubator containing 5% CO2 in the air. After incubation, cells were treated with DMSO (0.1%) as a control vehicle and the indicated concentration of AZD1480 for 2 h. Cells were fixed with 4% paraformaldehyde solution, permeabilized with Triton X-100 (0.2%), blocked with bovine serum albumin (BSA, Gibco, Waltham, MA, USA) and incubated with a primary antibody against FOXO3 followed by Alexa 594-conjugated anti-mouse secondary antibody (Invitrogen, Carlsbad, CA USA). After counterstaining with DAPI, fluorescence images were captured with a confocal microscope (Zeiss LSM510 microscope, Carl Zeiss, Jena, Germany).
Alexa 594 conjugated anti mouse secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 594-conjugated anti-mouse secondary antibody is a reagent used in immunoassays and other fluorescence-based applications. It is designed to detect and bind to primary antibodies raised against mouse antigens, allowing for the visualization and identification of target molecules or cells in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Lsm 510 microscope, by Zeiss (1 mentions)
Sucrose, by Fujifilm (1 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Mouse anti ha antibody, by Merck Group (1 mentions)
Zen software, by Zeiss (1 mentions)
4 protocols using alexa 594 conjugated anti mouse secondary antibody
1
Subcellular Localization of FOXO3 by Immunofluorescence
2
Immunofluorescence Localization of Luman in Mouse GCs
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Mouse GCs were fixed in 4% formaldehyde for 30 min and permeabilized in 0.2% Triton X-100 for 15 min at room temperature. After being blocked with 1% BSA in PBS, cover slips were incubated with an anti-Luman primary antibody (1:200 dilution; prepared by our laboratory), followed by incubation with an Alexa594-conjugated anti-mouse secondary antibody (1:300 dilution; Invitrogen, A31572) for 1 h at 37°C. Nuclei were stained with 4′,6-diamidino-2-phenylinole (DAPI) (Beyotime Co. Ltd) for 10 min. Images were captured with a digital camera under a Nikon epifluorescence microscope (Eclipse 80; Nikon, Tokyo, Japan).
3
Chondrocyte Identification and Actin Dynamics
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After 2 weeks of culture, pellets were fixed with 10% formaldehyde (Sigma-Aldrich) and dehydrated with 10% sucrose (WAKO chemical, Japan) in PBS for an hour. Frozen sections were prepared, blocked by 3.0% BSA (Gibco) in PBS, followed by staining with anti-collagen type II (1:100, Daiichi Fine Chemical, Japan) at 4°C, overnight. Samples were washed with PBS and stained with Alexa 594-conjugated anti-mouse secondary antibody (ThermoFisher Scientific, USA). Nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI).
To evaluate the effect of adding ROCKi on stress fiber formation, cultured NP cells in the ROCKi-treated and non-treated groups were evaluated by F-actin staining. Cells cultured up to P2 were seeded in a chamber slide (2 cm2) at a concentration of 5.0 × 103 cells/ml. The next day, cells were fixed with 10% formalin, and then, actin filaments were visualized by Alexa Fluor 594 phalloidin (ThermoFisher Scientific, USA) staining.
To evaluate the effect of adding ROCKi on stress fiber formation, cultured NP cells in the ROCKi-treated and non-treated groups were evaluated by F-actin staining. Cells cultured up to P2 were seeded in a chamber slide (2 cm2) at a concentration of 5.0 × 103 cells/ml. The next day, cells were fixed with 10% formalin, and then, actin filaments were visualized by Alexa Fluor 594 phalloidin (ThermoFisher Scientific, USA) staining.
4
Immunofluorescent Labeling of Kir2.1 Channels
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Cells expressing extracellular HA-tag on Kir2.1-GFP were fixed in 1% paraformaldehyde in phosphate buffer saline (PBS) for 10 min to retain GFP fluorescence, without permeabilization, then blocked with CAS blocking agent (Thermo Fisher Scientific) for 30 min at room temperature. Cells were incubated with mouse anti-HA antibody (Millipore, Burlington, MA, USA), washed with PBS, and incubated with Alexa-594 conjugated anti-mouse secondary antibody (ThermoFisher Scientific). Cell nuclei were counterstained with DAPI. Images were captured with an LSM 700 confocal microscope using Zen software (ZEISS, Oberkochen, Germany). Three channels (blue, green, red) were acquired sequentially.
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