SMSCs were seeded on glass coverslips in 24-well plates and treated as the experiment group processing. After another 24 hours, the chondrogenic induction cells were fixed and subsequently treated with 0.3% Triton X-100 (MP Biomedicals, USA). After washed with PBS, the cells were then incubated in 5% BSA. Then, the cells were incubated with microtubule-associated protein light chain 3 (LC3) rabbit monoclonal antibody (1 : 100; Abcam, USA) overnight at 4°C. The negative control group was treated with 1% BSA instead of the primary antibody. The FITC-conjugated goat antirabbit IgG (1 : 100; Proteintech, USA) was added for 1 hour and then incubated with DAPI staining solution (Leagene, China) for 15 min. The coverslip was dried and sealed with antifluorescence quenching agent (Beyotime, China). The cells were observed under laser confocal microscopy (FV10i, Olympus, Japan).
Anti fluorescence quenching agent
Manufactured by Beyotime
Sourced in China
The anti-fluorescence quenching agent is a chemical compound designed to reduce or eliminate the process of fluorescence quenching. Fluorescence quenching occurs when the intensity of fluorescence is decreased due to various factors, such as interactions with other molecules or the environment. The anti-fluorescence quenching agent is used to maintain the integrity and efficiency of fluorescence-based analytical techniques and measurements.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (4 mentions)
Eclipse 80i, by Nikon (2 mentions)
Paraformaldehyde (pfa), by Solarbio (2 mentions)
Triton x 100, by MP Biomedicals (1 mentions)
Fitc conjugated goat anti rabbit igg, by Proteintech (1 mentions)
Fv10i, by Olympus (1 mentions)
Dapi staining solution, by Leagene (1 mentions)
Rhodamine 123, by Beyotime (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Transfection reagent, by Roche (1 mentions)
Reverse transcription kit, by Promega (1 mentions)
Cellsens standard 1, by Olympus (1 mentions)
Normal donkey serum (nds), by Merck Group (1 mentions)
Apoptag peroxidase in situ apoptosis detection kit, by Merck Group (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Antibodies against p stat3 and stat3, by Cell Signaling Technology (1 mentions)
Nih elements system, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Goat serum, by Boster Bio (1 mentions)
20 protocols using anti fluorescence quenching agent
1
Chondrogenic Induction Cell Autophagy
2
Cell Labeling with Rhodamine123
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The cells were spread in 6-well plates containing slides and grouped and treated in the same way as before. After the treatment, the 6-well plates containing slides were rinsed with PBS three times. The cells were marked with Rhodamine123 (Beyotime, Shanghai, China) in a dark environment for a few minutes [55 (link)]. The dye solution in the hole was discarded, and the 6-well plate was washed with PBS three times. The slide was taken out and placed on a glass support containing an anti-fluorescence quenching agent (Beyotime, Shanghai, China) and photographed under a fluorescence microscope (Olympus, Tokyo, Japan).
3
Cell Viability Assay using Hoechst and PI
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Cells were seeded into 24-well plates with 50,000 cells per well. After the cells were treated according to the experimental design, the cells were washed three times with PBS. Then, 5 µl Hoechst staining solution was added, followed by 5 µl PI stain. The cells were mixed and incubated in an ice bath at 4˚C for 20-30 min. The cells were carefully washed three times with PBS and an anti-fluorescence quenching agent (Beyotime Institute of Biotechnology) was added. Cells were observed under a fluorescence microscope at x200 magnification and images captured. To calculate the percentage of PI-positive cells, the number of PI-positive cells was divided by the number of Hoechst-positive cells. Three random fields were chosen for this calculation.
4
Silkworm Strain Dazao Gene Expression
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The silkworm strain Dazao, used in this study, was provided by the State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China. BmE cell line was derived from the embryonic cells of the silkworm and reserved in our laboratory. The plasmid DNA extraction kit was purchased from TransGene (Beijing, China). The reverse transcription kit was obtained from Promega (Madison, WI, USA). The transfection reagent was a product of Roche (Basel, Switzerland). The specific primers were synthesized by Sangon Biotech (Shanghai, China). Polyoxymethylene was obtained from Sangon Biotech (Shanghai, China). Anti-fluorescence quenching agent and DAPI were obtained from Beyotime (Beijing, China). The prokaryotic expression plasmid pSKB2 was a gift from Prof. Xuewu Zhang (University of Texas Southwestern Medical Center at Dallas, TX, USA) and stored in our laboratory. The restriction enzymes were products of Takara (Tokyo, Japan).
5
RANKL-induced Osteoclast Differentiation
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RAW 264.7 cells were seeded at the density of 1,000 cells per well into 96-well plates and added 50 ng/mL RANKL to induce the osteoclast differentiation. Aucubin with various concentrations were added, and the TRAP staining kit (Cosmo bio company, Japan, cat: AK04F) was performed according to the manufacturer's instructions. The images were taken by Olympus microscope (Olympus cellSens standard1.18, Olympus, Japan) and TRAP positive multinucleated cells with three or more nuclei were regarded as Osteoclast-like cells. As for the F-actin staining, the cells were fixed with 4% paraformaldehyde (Biosharp, LOT 1810898) and washed with PBS three times. Then, cells were treated with permeabilized with 0.1% Triton X-100 for 5 min and stained with Rhodamine-Phalloidin (Biorigin, Beijing, BN10063) for 20 min. Subsequently, cell nuclei were stained with DAPI for 5 min and then mounted in antifluorescence quenching agent (Beyotime, Shanghai, China). The images were taken by Olympus microscope (Olympus cellSens standard1.18, Olympus, Japan).
6
Immunofluorescence Analysis of Epithelial-Mesenchymal Transition Markers
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The expressions of E-cadherin, α-SMA, BMP-7 and USAG-1 in NRK-52E cells were analyzed by immunofluorescence staining. Cells (2×104 cells/ml) were cultured on glass coverslips and treated with TGF-β1 with or without 1 µg/ml ART for 48 h. Following treatment for 48 h, cells in the basement layer were washed three times with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Following three extensive washes with PBS, the cells were treated with 0.5% Triton X-100 for 10 min and blocked with 2% normal donkey serum (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature, and subsequently incubated with primary antibodies (1:200) at 4°C overnight. Cells were washed with PBS for 3–5 min and incubated with goat anti-rabbit and rabbit anti-goat secondary antibodies (Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) at a dilution of 1:200 for 2 h. Following washing in PBS, cells were stained with DAPI (Beyotime Institute of Biotechnology) at room temperature to visualize the nuclei. Following washing in PBS, the device was mounted with anti-fluorescence quenching agent (Beyotime Institute of Biotechnology) and cover-slipped; digital images were captured using an inverted fluorescent microscope (magnification, ×400).
7
Apoptosis Analysis of Tumor Tissues
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Apoptosis was analyzed using the ApopTag Peroxidase in situ Apoptosis Detection kit (EMD Millipore). The tumor tissues were fixed in 4% paraformaldehyde at 4°C for 24 h, paraffin-embedded and sectioned (thickness, 3 µm). Tissue sections were deparaffinized and rehydrated and then incubated for 15-30 min at room temperature with 20 µg/ml proteinase K working solution (Beyotime Institute of Biotechnology). Subsequently, the TUNEL reaction mixture was added to the tumor sections. Following incubation in a humidified container for 2 h, the sections were mounted using anti-fluorescence quenching agent (Beyotime Institute of Biotechnology) and five random fields per slide were observed under a fluorescent microscope (magnification, ×200; Olympus Corporation).
8
NFV Induces Apoptosis via STAT3 Inhibition
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NFV was obtained from Shanghai Beilang Biotechnology Co., Ltd. (Shanghai, China).
NFV was dissolved in dimethylsulfoxide (DMSO). Antibodies against Bcl-xL, Bcl-2,
Cle-poly (ADP-ribose) polymerase (PARP), as well as horseradish
peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG, were
purchased from ythxbio (Beijing, China). Antibodies against p-STAT3 and STAT3
were purchased from Cell Signaling Technology (Danvers, MA, USA). DMSO and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were
purchased from Aladdin (Shanghai, China). The Annexin V-fluorescein
isothiocyanate/propidium iodide (PI) double staining apoptosis detection kit was
purchased from Shenzhen Kuyuan Biotechnology Co., Ltd. (Shenzhen, China).
Hoechst 33342 was purchased from Shanghai Wanlei Biology Co., Ltd. (Shanghai,
China) Anti-fluorescence quenching agent was purchased from Beyotime Institute
of Biotechnology (Beijing, China). Small-interfering RNA (siRNA) targeting STAT3
and the scrambled control were purchased from RIBOBIO (Suzhou, China).
Lipofectamine® RNAIMAX reagent was purchased from Thermo Fisher Scientific
(Waltham, MA, USA).
NFV was dissolved in dimethylsulfoxide (DMSO). Antibodies against Bcl-xL, Bcl-2,
Cle-poly (ADP-ribose) polymerase (PARP), as well as horseradish
peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG, were
purchased from ythxbio (Beijing, China). Antibodies against p-STAT3 and STAT3
were purchased from Cell Signaling Technology (Danvers, MA, USA). DMSO and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were
purchased from Aladdin (Shanghai, China). The Annexin V-fluorescein
isothiocyanate/propidium iodide (PI) double staining apoptosis detection kit was
purchased from Shenzhen Kuyuan Biotechnology Co., Ltd. (Shenzhen, China).
Hoechst 33342 was purchased from Shanghai Wanlei Biology Co., Ltd. (Shanghai,
China) Anti-fluorescence quenching agent was purchased from Beyotime Institute
of Biotechnology (Beijing, China). Small-interfering RNA (siRNA) targeting STAT3
and the scrambled control were purchased from RIBOBIO (Suzhou, China).
Lipofectamine® RNAIMAX reagent was purchased from Thermo Fisher Scientific
(Waltham, MA, USA).
9
Immunofluorescence Staining of Protozoa
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C. irritans theronts or T. thermophila were added dropwise to a slide and fixed with 4% paraformaldehyde (PFA) at 28°C for 15 min. The slides were washed and blocked with 10% goat serum at 37°C for 1 h. For detection of cross-reactive antigens, the slides were firstly incubated with grouper anti-serum (1:100 dilution) followed by incubation with anti-grouper IgM mAb and Alexa 488–conjugated anti-mouse IgG (1 μg/ml, CST). For tubulin detection, the slides were incubated with anti–rTt-tubulin pAb (1 μg/ml) followed by detection with Alexa 488–conjugated anti-rabbit IgG (1 μg/ml, CST). All slides were stained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen) and mounted in anti-fluorescence quenching agent (Beyotime). All slides were photographed using a NIH-Elements System (Nikon).
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C. irritans theronts or T. thermophila were added dropwise to a slide and fixed with 4% paraformaldehyde (PFA) at 28°C for 15 min. The slides were washed and blocked with 10% goat serum at 37°C for 1 h. For detection of cross-reactive antigens, the slides were firstly incubated with grouper anti-serum (1:100 dilution) followed by incubation with anti-grouper IgM mAb and Alexa 488–conjugated anti-mouse IgG (1 μg/ml, CST). For tubulin detection, the slides were incubated with anti–rTt-tubulin pAb (1 μg/ml) followed by detection with Alexa 488–conjugated anti-rabbit IgG (1 μg/ml, CST). All slides were stained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen) and mounted in anti-fluorescence quenching agent (Beyotime). All slides were photographed using a NIH-Elements System (Nikon).
10
Immunofluorescence Staining of Dental Progenitor Cells
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The third generation of DPCs was plated on cover glass (WHB, China) in 6-well plates for culturing 4 days. Removed the basal medium and rinsed for three times using phosphate buffer solution (PBS). And then fixed with 4% paraformaldehyde (Solarbio, #P1110, China) for 30 min in room temperature and washed with PBS for three times, permeabilized with 0.5% Trixton X-100 for 15 min in room temperature and washed with PBS for three times, blocked with goat serum (BOSTER, #12C09A, China) for 30 min and incubated with α-SMA antibody (BOSTER, #BM0002, China) /or Vimentin antibody (BOSTER, #BM0135, China) /or Wnt10b antibody (orb97574, biorbyt, U.S.A.) overnight at 4°C in wet box [18–20 (link)]. For immunofluorescence staining, we used SABC-FITC SP kit (BOSTER, #SA1062, China). The DPCs were incubated with goat anti mouse IgG secondary antibody for 30 min at 37°C and then added SABC-FITC for incubating at 37°C in darkness for 30 min after washing with PBS. Counter-staining with DAPI and mounting with anti-fluorescence quenching agent (Beyotime, #P0128, China). The fluorescence signals were observed by using a fluorescence microscope (Nikon ECLIPSE 80i, Japan).
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