A549 cells transfected with specific plasmids were seeded in 24-well plates at the indicated time points before immunofluorescence staining. The cells were washed in PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. After cells were washed with PBS, the cells were permeabilized using 0.25% Triton-X-100 for 5 min at room temperature and blocked in 10% donkey serum (BeyotimeInstitute of Biotechnology; Nanjing, China) for 30 min. The cells were subsequently incubated with primary antibodies against β-catenin (1:100; cat. no. ab16051; Abcam) overnight at 4°C. The following day, cells were washed in PBS and then incubated at room temperature for 1 h with a fluorescent-labeled secondary antibody (1:200; cat. no. A0562; BeyotimeInstitute of Biotechnology), followed by incubation with DAPI (1:1,000; cat. no. C1002; BeyotimeInstitute of Biotechnology). Images were captured under a confocal microscope.
Donkey serum
Manufactured by Beyotime
Sourced in China
Donkey serum is a biological fluid obtained from the blood of donkeys. It contains a variety of proteins, enzymes, and other biomolecules that are present in the donkey's circulatory system. The exact composition and properties of donkey serum can vary depending on the source and processing methods.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (3 mentions)
Triton x 100, by Beyotime (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ab185966, by Abcam (1 mentions)
A11005, by Thermo Fisher Scientific (1 mentions)
Ab34712, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Fluorescent labeled secondary antibody, by Beyotime (1 mentions)
Smad3, by Abcam (1 mentions)
Fitc conjugated anti rabbit igg, by Abcam (1 mentions)
Anti mouse igg, by BioLegend (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
7 protocols using donkey serum
1
Immunofluorescence Staining of β-Catenin in A549 Cells
2
Immunofluorescence Visualization of Chondrocyte Markers
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TNF-α- and IL-1β-treated rat chondrocytes were rinsed in PBS and then fixed with 10% neutral-buffered formalin for 30 min at 21 °C. Cells were incubated with Triton X-100 (Beyotime Biotechnology, Beijing, China) for 10 min to penetrate the cell membrane and donkey serum (Beyotime Biotechnology) for 1 h to block nonspecific binding sites. Cultured cells were incubated with primary antibodies against type II collagen (ab34712; Abcam; 1:100), SOX9 (ab185966; Abcam; 1:100), and LRP3 (sc-373736; Santa Cruz Biotechnology, USA; 1:50) at 4 °C overnight. The cells were then washed with PBS three times and incubated with Alexa Fluor 488-conjugated goat anti-rabbit (A-11008, Thermo Fisher Scientific; 1:200) or Alexa Fluor 594-conjugated goat anti-mouse (A-11005, Thermo Fisher Scientific; 1:200) secondary antibodies for 1 h at 21 °C. Nuclei were stained with DAPI (Beyotime Institute of Biotechnology, Jiangsu, China) for 10 min. Finally, the samples were rinsed with PBS and visualized using a confocal microscope (Olympus Life Science, Tokyo, Japan).
3
Immunofluorescence Analysis of SMAD3 in HeLa Cells
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HeLa cells transfected with specific plasmids were seeded in 24‐well plates at the indicated time points before immunofluorescence staining. Cells were washed in PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature. After cells were washed with PBS, the cells were permeabilized using 0.25% Triton‐X‐100 for 5 minutes at room temperature and blocked in 10% donkey serum (Beyotime Biotechnology, Nanjing, China) for 30 minutes. The cells were subsequently incubated with primary antibodies against SMAD3 (1:200; Abcam, Cambridge, MA, USA) overnight at 4°C. The following day, cells were washed in PBS and then incubated at room temperature for 1 hour with a fluorescent‐labeled secondary antibody (1:200; Beyotime Biotechnology), followed by incubation with DAPI (1:1000; Beyotime Biotechnology). Images were captured under a confocal microscope.
4
Immunofluorescence Analysis of Chondrocyte Markers
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Cultured chondrocytes were rinsed in PBS, and then fixed with 10% neutral buffered formalin for 30 min at room temperature. Triton X-100 (Beyotime Biotechnology) was used to penetrate the cell membrane for 5 min, and donkey serum (Beyotime Biotechnology) was applied to block nonspecific binding sites for 1 h. Cultured cells were incubated with primary antibodies against type II collagen (Abcam; 1:200) and SOD3 (Santa Cruz Biotechnology; 1:100) at 4 °C overnight. The cells were then washed with PBS three times, and then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (Abcam; 1:1000) or anti-mouse IgG (Biolegend, USA; 1:200) for 1 h. Nuclei were stained with Hoechst 33258 for 5 min. Finally, the samples were rinsed with PBS and visualized using confocal microscopy. LAS_X software (Flexera software LLC) was applied to quantify the fluorescence intensity of rat chondrocytes from at least 6 images.
5
Immunofluorescence Assay of p65 Translocation
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Cells transfected with the indicated plasmids before IF assay. Forty-eight hours later, the cells were washed with 1×PBS, fixed in 4% paraformaldehyde for 30 min and blocked with 10% donkey serum (Beyotime Institute of Biotechnology; Nanjing, China) for 30 min at room temperature. The primary antibody against p65 was incubated overnight at 4°C. The fluorescein isothiocyanate (FITC)-conjugated secondary antibody was added and incubated for 2 h, followed by incubation with DAPI. Fluorescence images were captured using a confocal microscope.
6
Investigating NF-κB Pathway Regulation
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4×104 A549 cells transfected with specific plasmids (1 µg pcDNA3, 1 µg pri-miR-505, 1 µg MAP3K3 or 0.5 µg pri-miR-505+0.5 µg MAP3K3) using Lipofectamine® 2000, according to the manufacturer's protocols were seeded with Dulbecco's modified Eagle's medium containing 10% FBS in 24-well plates for 48 h and then for immunofluorescence staining. The cells were washed in PBS three times and fixed with 4% paraformaldehyde for 30 min at room temperature. Following cells being washed with PBS three times, the cells were permeabilized using 0.1% Triton-X-100 for 10 min at room temperature and blocked in 10% donkey serum (Beyotime Institute of Biotechnology) for 30 min at room temperature. The cells were subsequently incubated with primary antibodies against p50 (cat. no. WL01917, 1:50) and p65 (cat. no. WL01980; 1:50) (both from Wanlei Co., Ltd.) overnight at 4°C. The following day, cells were washed in PBS three times and then incubated at room temperature for 1 h with a goat anti-rabbit IgG H&L Alexa Fluor® 488 (cat. no. ab150077; 1:100; Abcam), followed by incubation with DAPI (1:1,000; Sigma-Aldrich; Merck KGaA) at room temperature. Images were captured under a confocal microscope (at ×1,000).
7
Immunohistochemistry of Frozen Sections
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Formalin-xed OCT-embedded frozen sections (40 µm thick) were permeabilized with 0.3% Triton X-100 (Beyotime Biotechnology Co., Ltd, ST797, Shanghai, China) in PBS, followed by blocking for 4 h in 0.3% Triton X-100 with 10% donkey serum (Beyotime Biotechnology Co., Ltd, A7039, Shanghai, China). The sections were incubated in the primary antibody diluted in 0.3% Triton X-100 with 10% donkey serum for 48 h at 4°C. After primary antibody incubation, the sections were washed in PBS and incubated in the secondary antibody diluted in 0.3% Triton X-100 with 10% donkey serum for 4h at room temperature (RT). Sections were washed three times in PBS and incubated in Hoechst 33342 (1:2,000; Thermo Fisher) for 15 min at RT for nuclear counterstaining. The sections were then mounted on microscope slides, sealed with clear nail polish, and stored at 4°C for preservation. The negative control sections were incubated with only secondary antibodies. There were six mice in each of the three groups. The antibody information is listed in Supplementary Tables 1 and2.
In our study, omission of the primary antibody and incubation of the sections only with secondary antibodies was used to con rm the speci city of the different secondary antibodies.
In our study, omission of the primary antibody and incubation of the sections only with secondary antibodies was used to con rm the speci city of the different secondary antibodies.
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