Purified EVs were mixed and incubated with PHK67 (MINI67, Sigma-Aldrich) for 5 min. The staining was stopped with the addition of 2 mL of 10% bovine serum albumin (BSA). Subsequently, EV-free serum medium was supplemented to 8.5 mL, and 1.5 mL of 0.971 M sucrose was slowly added from the bottom of the test tube to ensure that PKH-67-EVs were on top of the sucrose solution. Following centrifugation, EV pellets were resuspended in PBS. Next, SH-SY5Y cells were cultured, the medium was renewed for 48 h, and added with PKH67-labeled EVs at 37 °C for 6 h. Later, the cells were fixed with 4% polytetrafluoroethylene for 15 min, stained with 0.1 g/mL DAPI (C1002, Beyotime, Nantong, China) for 5 min, and then SH-SY5Y cells were placed in a fluorescence microscope (Olympus, Tokyo, Japan) to detect the fluorescence expression.
Dapi c1002
Manufactured by Beyotime
Sourced in China
DAPI (C1002) is a fluorescent dye used in molecular biology and cell biology for the staining of cellular nuclei. It binds strongly to DNA and emits blue fluorescence when excited by ultraviolet light.
Automatically generated - may contain errors
Lab products found in correlation
Transwell chamber, by Corning (3 mentions)
Tcs sp5 2, by Leica (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Ki67 antibody, by Abcam (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Fv1000, by Olympus (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Phk67, by Merck Group (1 mentions)
Alexa fluor 594 affinipure donkey anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Fv1000 spectral confocal microscope, by Olympus (1 mentions)
Flowview acquisition software, by Olympus (1 mentions)
Ab178846, by Abcam (1 mentions)
Alexa fluor 488 affinipure goat anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
Fitc conjugated secondary antibody, by Boster Bio (1 mentions)
Bx53 fluorescent microscope, by Olympus (1 mentions)
Sa00013 1, by Proteintech (1 mentions)
Sa00013 2, by Proteintech (1 mentions)
Cell light edu fluorescence detection kit, by RiboBio (1 mentions)
Fsx100, by Olympus (1 mentions)
50 protocols using dapi c1002
1
Fluorescent Labeling of EVs for Cellular Uptake
2
Immunofluorescence analysis of microglia
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HAPI microglia cells were fixed in 4% PFA for 10 min and rinsed with PBS three times. The samples were permeabilized with 0.03% Triton X-100 for 10 min, and then blocked using 1% (w/v) BSA in PBS for 30 min and incubated with primary antibodies CD40 (1:10, sc-514493, Santa Cruz Biotechnology, Santa Cruz-CA, USA) and Iba-1 (1:200, ab178846, Abcam) at 4 °C overnight. After washing off the primary antibody with PBS, secondary antibodies Alexa Fluor® 488 AffiniPure Goat Anti-Mouse IgG (H+L) (1:100, 115-545-003, Jackson ImmunoResearch, West Grove, PA, USA) and Alexa Fluor® 594 AffiniPure Donkey Anti-Rabbit IgG (H+L) (1:100, 711-585-152, Jackson ImmunoResearch, West Grove, PA, USA) were applied at 37 °C in the dark for 30 min. DAPI (C1002, Beyotime, Shanghai, China) was used to counterstain nuclei for 10 min in the dark at room temperature. Images were acquired at room temperature with an Olympus FV1000 spectral confocal microscope (Olympus Corporation, Tokyo, Japan) with an UPLFLN 40× objective lens (NA 1.30) and Olympus FLOWVIEW acquisition software. To quantify fluorescence, the fluorescence intensity under different transfection conditions were analyzed by Image J software (http://rsb.info.nih.gov/ij ).
3
Immunofluorescence Analysis of AdipoR1 and AdipoR2
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RWPE1 and WPMY1 cells were plated on Fisherbrand Coverglass (6-well, Thermo Fisher, USA) at a density of 5 × 105 per well. After regular incubation for 24 h, 4% paraformaldehyde was used to fix the cells for 20 min. Then, the cells were blocked with goat serum and incubated with primary antibodies against AdipoR1 (1:200) and AdipoR2 (1:200) overnight at 4 °C. Finally, the slides were incubated with a FITC conjugated secondary antibody (1:100, Boster, Wuhan, China) for 1 h at 37 °C. The nuclei were counterstained with DAPI (C1002, Beyotime Biotechnology, China). The slides were photographed under a BX53 fluorescent microscope (Olympus).
4
Immunocytochemistry for Cell Imaging
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Cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with Triton X-100 (1139ML100, Biofroxx) for 20 minutes, blocked with 1% BSA (4240GR100, Biofroxx) for 30 minutes, and incubated overnight with specific primary antibody. Thereafter, probe cells with conjugated goat anti-mouse IgG (H + L) (SA00013-1, Proteintech) or rabbit IgG (H + L) (SA00013-2, Proteintech) at room temperature in the dark for one hour. Counterstain cell nuclei with DAPI (C1002, Beyotime). Then observe cells under a fluorescent microscope.
5
Immunofluorescence of Fixed and Permeabilized Cells
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Huh7 cells were washed and fixed in 4% paraformaldehyde for 10 min at room temperature. Fixed cells were then permeabilized with 0.1% Triton X-100 in PBS for 30 min at room temperature. After being blocked in PBS containing 20% goat serum, cells were incubated with the indicated primary antibodies for 1 h, rinsed with PBS, then incubated with AlexaFluor-tagged secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. Cells were then stained with DAPI (C1002, Beyotime, Shanghai, China) to show the nuclei.
6
EdU Proliferation Assay Protocol
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A Cell-Light EdU fluorescence detection kit (Guangzhou RiboBio Co., Ltd.) was used to detect EdU-positive cells according to the manufacturer's instructions. The cells were seeded into 96-well plates (5,000 cells/well), added with 100 μl medium containing 50 μmol/l EdU, and cultured at 37°C with 5% CO2 for 2 h. The cells were then washed with PBS 3 times and each well was supplemented with 100 μl PBS solution containing 4% paraformaldehyde to fix the cells at room temperature for 30 min. Following PBS washing, each well was supplemented with 100 μl PBS solution containing 0.5% TritonX-100 to increase membrane permeability. Following PBS washing again, each well was added with 100 μl Apollo® staining reaction solution (provided with the kit) and cultured at room temperature in the dark for 30 min. The staining reaction solution was discarded and each well was supplemented with 100 μl DAPI (C1002, Beyotime Institute of Biotechnology, Inc.) at room temperature in the dark for 30 min. Following PBS washing, the staining effect was observed under fluorescence microscope (FSX100, Olympus Corporation) and images were collected. The images were analyzed using Olympus stream system. The total number of nuclei (blue) and proliferating cells (red) were counted respectively. EdU-positive cell rate=proliferating cells/total cells ×100%. The experiment was repeated three times.
7
Autophagy Regulation in Cell Assays
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Opti-MEM (31985088) was purchased from Gibco. MitoTracker Red CMXRos (40740ES50) was purchased from YEASEN. DAPI (C1002) and N-acetylcysteine (S0077) were purchased from Beyotime. 3-methyladenine (3-MA) (S2767) and bafilomycin A1 (Baf-A1) (S1413) were purchased from Selleck Chemicals. CCCP (C2759) (SAB5701328) was purchased from Sigma-Aldrich. Anti-Occludin (91131S), Claudin-1 (13255S), GAPDH (5174S), Parkin(2132S), and β-actin (3700S) were purchased from Cell Signaling Technology. Anti-P62 (T55546), ATG5 (T55766), and LC3B primary antibodies were purchased from Abmart.
8
Immunofluorescence Staining of Tissue and Cells
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Immunofluorescence staining was performed on tissue and cell samples. For tissue samples, slides were deparaffinized, rehydrated, and treated with sodium citrate buffer for antigen retrieval. Slides were incubated with 3% goat serum for 30 min followed by primary antibodies. Cell samples were fixed in poly‐l ‐lysine polylysine (P2100, Solarbio, China), followed by permeabilization and non‐specific binding site blocking. Samples were treated at 4°C overnight with the following primary antibodies: rabbit anti‐MPO antibody, mouse anti‐NE antibody, goat anti‐shp2 antibody (ab9214, Abcam, USA), rabbit anti‐pad4 antibody (ab214810, Abcam, USA), and rabbit anti‐histone H3 (citrulline R2) antibody (ab174992, Abcam, USA). The next day, Samples were incubated with an Alexa fluorescein‐labeled secondary antibody for 2 h. DAPI (C1002, Beyotime, China) was used to stain cell nuclei. Then, samples were imaged by an inverted confocal microscope (LSM880 with airyscan, Carl Zeiss, Germany).
9
Immunofluorescence Analysis of Sirt3 Expression
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Cells were fixed in 4% paraformaldehyde (8009628, Sinopharm Chemical Reagent) or 15 min, permeabilized in 0.5% Triton X‐100 (DH351‐2, Beijing Dingguo Changsheng Biotechnology) in PBS for 5 min, blocked with 4% goat serum in PBS for 60 min, and incubated with rabbit monoclonal anti‐Sirt3 (Ab86671, Abcam) overnight at 4°C. DyLight 488‐conjugated goat anti‐rabbit (GAR4882, Multi Sciences) was used as a secondary antibody. Samples were stained with DAPI (C1002, Beyotime) before photographed on a fluorescence microscope (FV1000MPE‐share).
10
Immunostaining of Autophagy Markers in A549 Cells
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A549 cells were washed with cold 1xPBS (Dingguo Bio, Beijing, China) and then fixed with 4% paraformaldehyde (Biosharp, Beijing, China) at room temperature for 30 minutes. After being washed thrice with cold 1xPBS (Dingguo Bio, Beijing, China), the cells were blocked in 10% BSA (Beyotime, Shanghai, China) at room temperature for 10 min. The cells were subsequently incubated with primary antibodies specific for LC3 (SRP01707, Saier Biotechnology, Tianjin, China) at 4°C overnight. The next day, the cells were washed with cold 1xPBS (Dingguo Bio, Beijing, China), after which they were incubated with fluorescence-conjugated secondary antibodies (A0562, Beyotime, Shanghai, China) at room temperature for 1 h, followed by DAPI (C1002, Beyotime, Shanghai, China) at room temperature for 5min. Images were captured under a confocal microscopy.
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