SCs and HUVECs were seeded in 6-well plates at a density of 3 × 105 cells per well. One Chi, Chi/PDA, or Chi/PDA-Ps conduit was placed into each well, and the cells were cultured for 5 days. The SCs and HUVECs were then gently rinsed with 37°C PBS and fixed with 4% paraformaldehyde for 15 minutes. The cell samples were permeabilized with 0.5% Triton X-100 (MilliporeSigma) at room temperature for 5 minutes. The SCs samples were incubated with rabbit anti-S100 antibody (1:400, MilliporeSigma, Cat# S2644, RRID: AB_477501) at 4°C overnight, and the HUVECs samples were incubated with fluorescein phalloidin (1:1000, MilliporeSigma) at room temperature for 1 hour. Alexa Fluor 594-conjugated goat anti-rabbit secondary antibodies (1:200, Abcam, Cambridge, UK, Cat# ab150080, RRID: AB_2650602) were incubated with the SC samples in the dark for 2 hours at room temperature. The samples were gently washed in PBS and incubated with 4′,6-diamidino-2-phenylindole dihydrochloride (1:1000, MilliporeSigma) at room temperature for 30 minutes. Finally, a fluorescence microscope (Leica) was used to image the cell samples.
Alexa fluor 594 conjugated goat anti rabbit secondary antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Alexa Fluor 594 conjugated goat anti-rabbit secondary antibody is a fluorescently-labeled antibody used to detect and visualize rabbit primary antibodies in various applications, such as immunofluorescence, flow cytometry, and Western blotting. The antibody is conjugated to the Alexa Fluor 594 dye, which emits a red fluorescent signal when excited by the appropriate wavelength of light.
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Lab products found in correlation
Tcs sp5 confocal laser scanning microscope, by Leica (3 mentions)
Triton x 100, by Merck Group (2 mentions)
γh2ax antibody, by Cell Signaling Technology (2 mentions)
Fluorescence microscope, by Leica (1 mentions)
Lsm 710 meta confocal laser scanning microscope, by Zeiss (1 mentions)
Zen 2.3 blue edition software, by Zeiss (1 mentions)
Glass coverslips, by Thomas Scientific (1 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (1 mentions)
Cell titer 96 aqueous one solution cell proliferation assay solution, by Promega (1 mentions)
6 protocols using alexa fluor 594 conjugated goat anti rabbit secondary antibody
1
Cellular Interactions with Biomaterial Conduits
2
P2X7 Receptor Immunocytochemistry in Neuro-2a Cells
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Neuro-2a cells on coverslips were washed once with PBS, fixed with cold 100% methanol for 5 min in an ice bath, and then washed (3 × 5 min) with PBS. For P2X7 receptor immunocytochemistry, cells were blocked with 10% bovine serum in PBS containing 0.1% Triton X-100 (1 h) and incubated with specific to P2X7 primary antibodies (1:1000; orb35965 Biorbyt Ltd., Cambridge, UK) overnight at 4 °C. Cells were washed with PBS (3 × 10 min) and incubated with Alexa Fluor 594-conjugated goat anti-rabbit secondary antibodies (1:200, Abcam plc, Cambridge, UK) for 2 h at room temperature. Cells were washed in PBS (3 × 10 min) before mounting. In control experiments, the primary antibody was omitted, and immunostaining was never observed. Neuro-2a cell fluorescence was studied using an LSM 710 META confocal laser scanning microscope (Carl Zeiss, Göttingen, Germany) equipped with an Ar laser with an effective power of 30 mW. The obtained images were analyzed using the ZEN 2.3 (blue edition) software (Carl Zeiss, Göttingen, Germany).
3
Quantifying DNA Damage via γ-H2AX Foci
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DNA damage was determined using immunofluorescence staining of γ-H2AX foci. U251 and LN229 cells were treated as described in 2.8. Cells were incubated with γ-H2AX antibody (Cell Signaling Technology; Danvers, MA, USA) and Alexa Fluor 594 conjugated goat anti-rabbit secondary antibody (Abcam; Cambridge, UK). Red dots corresponding to γ-H2AX were counted under a Leica TCS SP5 Confocal Laser Scanning Microscope (Leica Microsystem).
4
Photocrosslinkable Gelatin Hydrogel for Liver Metabolism
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Gelatin from porcine skin (type-A, 300 bloom), methacrylic anhydride, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), Opti-MEM I reduced serum medium (Opti-MEM), fetal bovine serum (FBS), goat serum, trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA), antibiotic-antimycotic solution stabilized (Anti-Anti, 100X), 4′,6-diamidino-2-phenylindole (DAPI), formalin (10% w/v), Live/Dead® Viability/Cytotoxicity Kit, Alexa Fluor® 594-phalloidin, dialysis membrane (Mw cutoff: 12–14 kDa) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The CellTiter 96® AQueous One Solution Cell Proliferation Assay solution was obtained from Promega (Madison, WI, USA). Mouse cytochrome P450 3A (CYP3A4/CYP3A5 monoclonal) antibody and Alexa Fluor® 594-conjugated goat anti-rabbit secondary antibody were purchased from Abcam (Cambridge, MA, USA). Ruthenium visible light photocrosslinking kit was purchased from Advanced BioMatrix (San Diego, CA, USA). Glass coverslips of 8 mm in diameter were obtained from Thomas Scientific (Swedesboro, NJ, USA). All other chemicals used in this study were obtained from Sigma-Aldrich unless otherwise mentioned.
5
Quantifying DNA Damage via γ-H2AX
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DNA damage was determined using immunofluorescence staining of γ-H2AX foci. Fadu cells treated with negative control siRNA, UVRAG siRNA, 4 Gy irradiation, UVRAG siRNA combining 4 Gy irradiation were incubated with γ-H2AX antibody (Cell Signaling Technology; Danvers, MA, USA) and Alexa Fluor 594 conjugated goat anti-rabbit secondary antibody (Abcam; Cambridge, UK). Red dots were counted under a Leica TCS SP5 Confocal Laser Scanning Microscope.
6
Lipid Peroxidation Visualization in Cancer Cells
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LN229 and U118 cells were treated with 1 μM FIN56 or DMSO for 24 h. Then cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and incubated with 4-Hydroxynonenal (4-HNE, a lipid peroxidation marker) primary antibody (Abcam; Cambridge, UK) and Alexa Fluor 594 conjugated goat anti-rabbit secondary antibody (Abcam). Images were taken under a Leica TCS SP5 Confocal Laser Scanning Microscope (Leica Microsystem).
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