The 131I-PLGA-rhTSH nanoparticles were mounted on glass slides and fixed with 4% paraformaldehyde (PFA) in 0.01 M PBS for 30 min at room temperature. Then, the samples were incubated with anti-TSHR primary antibodies (Abcam, Cambridge, UK) or anti-cleaved caspase-3 primary antibodies (Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. On the following day, the samples were immersed in Alexa Fluor® 555-conjugated secondary antibody (1:100, Beyotime, Shanghai, China) for 2 h at room temperature after being washed with PBS. Subsequently, 4′-6-diami-dino-2-phenylindole (DAPI; Beyotime, Shanghai, China) was used to counterstain cell nuclei for 10 min at room temperature. The images were captured with a fluorescence microscope (Carl Zeiss, Weimar, Germany) and analyzed with ImageJ software (ImageJ 1.8, NIH, USA).
Alexa fluor 555 conjugated secondary antibody
Manufactured by Beyotime
Sourced in China
Alexa Fluor 555-conjugated secondary antibody is a fluorescently labeled antibody that can be used to detect and visualize target proteins in various biological applications, such as immunofluorescence, flow cytometry, and Western blotting. The Alexa Fluor 555 dye provides a bright, photostable signal that can be detected using standard fluorescence microscopy or flow cytometry equipment.
Automatically generated - may contain errors
Lab products found in correlation
Anti γ h2ax primary antibody, by Cell Signaling Technology (2 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Confocal uorescence microscope, by Leica (1 mentions)
Confocal fluorescence microscope, by Leica (1 mentions)
6 protocols using alexa fluor 555 conjugated secondary antibody
1
Immunofluorescence Analysis of TSHR and Caspase-3 in Thyroid Cells
2
Immunofluorescence Analysis of TGM2 in HCC
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Expression of TGM2 in HCC cells was determined by immunofluorescence30 (link). HCC cells were grown on glass cover slips to 20–30% confluency and then fixed, permeabilized, blocked, and incubated with TGM2 antibody overnight at 4 °C. The slides were subsequently washed and incubated with Alexa fluor 555-conjugated secondary antibody (Beyotime Biotechnology). To visualise nuclear details, the cells were counterstained using 4′-6-diamidino-2-phenylindole (DAPI), and fluorescence microscopic examination (Olympus) was performed.
3
Quantifying DNA Damage Response
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A total of 5 × 104 cells were seeded into a confocal laser dish 1 day before irradiation with 8 Gy. Four hours post irradiation, cells were fixed with 4% paraformaldehyde at room temperature for 30 min and permeabilized with 0.1% Triton X-100 for 2 h. Cells were then blocked with 5% BSA for 90 min and washed with PBS. After incubation with the anti-γ-H2AX primary antibody (1:400; Cell Signaling Technology) overnight at 4 °C, cells were washed with PBS and incubated with an Alexa Fluor 555-conjugated secondary antibody (Beyotime) for 90 min. Cells were washed with PBS, treated with DAPI staining solution for 20 min and observed using a confocal fluorescence microscope (Leica).
4
Visualizing Nrf2 Translocation in Osteoclasts
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RAW 264.7 cells were cultured in 96-well plates, followed by treatment as described in Sect. “TRAP staining and TRAP activity measurement ”. After 5 d, the cells were washed with PBS, after washing cells were fixed in 4% paraformaldehyde (PFA) for 15 min, washed with PBS three times, permeabilized with 0.1% TritonX-100 for 15 min, and stained with rhodamine-phalloidin in the dark for 1 h. Nuclei were stained with DAPI for 10 min at room temperature. Finally, fluorescence images were obtained using a fluorescence microscope (Leica, Germany).
We performed Nrf2 immunofluorescence staining and nuclear translocation assays. RAW264.7 cells were seeded into 96-well plates and allowed to adhere overnight. The next day, the cells were treated with ISO (100 μM) for 2 h and then stimulated with RANKL for 30 min. After fixation with 4% PFA, the cells were blocked with 2% bovine serum albumin (diluted in PBS) for 1 h and incubated with an anti-Nrf2 antibody at 4 °C overnight. The cells were then incubated with Alexa Fluor 555-conjugated secondary antibody (Beyotime, China) for 2 h in the dark. After 3 times washing with PBS, the cells were stained with DAPI, and the final immunofluorescence images were obtained.
We performed Nrf2 immunofluorescence staining and nuclear translocation assays. RAW264.7 cells were seeded into 96-well plates and allowed to adhere overnight. The next day, the cells were treated with ISO (100 μM) for 2 h and then stimulated with RANKL for 30 min. After fixation with 4% PFA, the cells were blocked with 2% bovine serum albumin (diluted in PBS) for 1 h and incubated with an anti-Nrf2 antibody at 4 °C overnight. The cells were then incubated with Alexa Fluor 555-conjugated secondary antibody (Beyotime, China) for 2 h in the dark. After 3 times washing with PBS, the cells were stained with DAPI, and the final immunofluorescence images were obtained.
5
Visualizing DNA Damage Response
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A total of 5 × 10 4 cells were seeded into a confocal laser dish one day before 8 Gy irradiation. Four hours post-irradiation, cells were xed in 4% paraformaldehyde at room temperature for 30 minutes and permeabilized in 0.1% Triton X-100 for 2 hours. Cells were then blocked with 5% BSA for 90 minutes and washed with PBS. After incubation with the primary antibody γ-H2AX (1:400; Cell Signalling Technology) overnight at 4℃, the cells were washed with PBS and incubated with an Alexa Fluor 555-conjugated secondary antibody (Beyotime) for 90 minutes. Cells were washed with PBS and treated with DAPI staining solution for 20 minutes and then observed using a confocal uorescence microscope (Leica).
6
Quantifying DNA Damage in Irradiated Cells
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A total of 5×10 4 cells were seeded into a confocal laser dish one day before irradiation with 8 Gy. Four hours post irradiation, cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes and permeabilized with 0.1% Triton X-100 for 2 hours. Cells were then blocked with 5% BSA for 90 minutes and washed with PBS. After incubation with the anti-γ-H2AX primary antibody (1:400; Cell Signaling Technology) overnight at 4 , cells were washed with PBS and incubated with an Alexa Fluor 555-conjugated secondary antibody (Beyotime) for 90 minutes. Cells were washed with PBS, treated with DAPI staining solution for 20 minutes and observed using a confocal fluorescence microscope (Leica).
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