The terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling (TUNEL) assay was performed to identify apoptotic cells using an in situ cell death detection (POD) kit (KeyGEN BioTECH, Jiangsu, China). After conditioned medium treatment, neurons cultured on coverslips in poly-D-lysine-coated six-well plates for 7 days were rinsed three times with PBS and fixed with freshly prepared 4% paraformaldehyde for 20 min at room temperature. Cells were blocked in 3% H2O2 with PBS for 15 min and then permeabilized in ice-cold 0.1% Triton X-100 with PBS for 10 min. TUNEL-positive (apoptotic) neurons were identified and counted according to the assay manufacturer's instructions. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Nantong, Jiangsu, China), and the number of TUNEL+-/DAPI+-neurons converted to a percentage of the total DAPI+-nuclei in four nonoverlapping fields per coverslip.
4 6 diamidino 2 phenylindole (dapi)
Manufactured by Beyotime
Sourced in China, United States, Germany, Japan, United Kingdom, Switzerland, Puerto Rico
DAPI is a fluorescent dye used in molecular biology and microscopy to stain and visualize DNA. It selectively binds to the minor groove of double-stranded DNA, emitting a blue fluorescence when excited by ultraviolet light. DAPI is commonly used for nuclear staining and counterstaining in various applications such as fluorescence microscopy and flow cytometry.
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Lab products found in correlation
Fluorescence microscope, by Olympus (154 mentions)
Triton x 100, by Beyotime (106 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (72 mentions)
Triton x 100, by Merck Group (67 mentions)
Fluorescence microscope, by Leica camera (62 mentions)
Paraformaldehyde (pfa), by Beyotime (60 mentions)
Antifade mounting medium, by Beyotime (59 mentions)
Triton x 100, by Solarbio (54 mentions)
Lsm 780, by Zeiss (52 mentions)
Lsm 710, by Zeiss (52 mentions)
Lsm 880, by Zeiss (50 mentions)
Fluorescence microscope, by Nikon (46 mentions)
Fv1000, by Olympus (39 mentions)
In situ cell death detection kit, by Roche (38 mentions)
Bovine serum albumin (bsa), by Beyotime (36 mentions)
Bovine serum albumin (bsa), by Merck Group (35 mentions)
Goat serum, by Beyotime (34 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (34 mentions)
Tcs sp8, by Leica camera (31 mentions)
Confocal laser scanning microscope, by Zeiss (30 mentions)
3 645 protocols using 4 6 diamidino 2 phenylindole (dapi)
1
Apoptosis Quantification in Neurons
2
Circulating Tumor Cell (CTC) Detection
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The detailed protocols for CTC detection were performed as previously described.17 (link) In brief, blood samples were harvested preoperatively for CTC detection. The samples were centrifuged (1500 rpm), then the upper clear supernatant was discarded and incubated with 30 µL of immuno nano-magnetic spheres for 30 minutes (Magnetic nanoparticles, ShengNa Industrial Limited Company, Shanghai, China; BaiHuiKang Biotechnology Co. Shanghai, China). Cells enriched by magnetic isolation were stained by adding 2 μL of diamidino-phenylindole(DAPI), 10 μL of CK-19-FITC and 10 μL of CD45 (DAPI, BiYunTian Biotechnology Co., China; Anti-CK-19, phycoerythrin (PE), Johnson & Johnson Medical Veridex Company, USA; Anti-CD-45-PE, Becton, Dickinson and Company, USA). Observation of CTCs was carried out after air-drying; the pictures with fluorescence staining were captured under a fluorescence microscope (Olympus B*61, USA) for the identification and counting of CTCs.
3
Immunofluorescence Analysis of NLRP3 and USP7 in OS Cells
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The U2OS cells were seeded to the cover slides coated petri dishes. The cover slides were fixed by 4% paraformaldehyde after incubation with phosphate buffered saline (PBS), E2 (100 nmol/L), Torin1 (1 μM/L), E2+ Torin1 treatment for 24 h. 0.5% Triton X-100 was added for cell permeability for 20 min. After blocking, the slides or or OS tissue sections were incubated with the primary antibodies, anti-ASC speck (1:200, Santa Cruz, CA, USA) at 4 °C overnight, respectively. Then the slides were incubated with Alexa Fluor 488-conjugated secondary antibody (1:400, Life Techonologies, USA) for 30 min following washed by PBS for three times. Finally, the slides were counterstained with DAPI (Beyotime, China) and analyzed with a fluorescence microscope (Leica, Wetzlar, Germany). We used 4% paraformaldehyde to fix the OS tissues or normal tissues. After blocking, the slides were incubated with Alexa Fluor 594 conjugated USP7 antibody (1:200, Novus Biologicals, CO, USA), Alexa Fluor 488 conjugated NLRP3 antibody (1:100, R&D Systems, USA) or ASC antibody (1:100, Cell Signaling Technology) at 4 °C for 60 min, then washed by PBS. Finally, we used DAPI (Beyotime, China) to counterstain the slides and used a confocal microscope (Leica, Wetzlar, Germany) to analyze and photograph.
4
Visualizing iNKT Cell Activation
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For α-GalCer stimulation, the sorted iNKT cells were seeded in a 96-well plate in DMEM (with 10% FBS), left unstimulated, or stimulated with α-GalCer (125 ng/ml) for 72 h. α-GalCer -stimulated iNTK cells were dropped on the poly-L-lysine slides, incubated for 30 min, fixed with 4% paraformaldehyde, and then permeabilized with 0.05% PB buffer. The unstimulated iNKT cells were incubated with a mouse anti-PLZF antibody (4 μg/ml, Santa Cruz Biotechnology) and AF488-Actin (Invitrogen) for 1 h, further stained with a Goat anti-mouse AF546 secondary antibody (1:400) for 30 min, and finally covered with 1.5 μg/ml DAPI (Beyotime). α-GalCer stimulated iNTK cells were incubated with rabbit-anti-FoxO-1 (Cell signaling technology) and AF488-Actin for 1 h, and further stained with a AF546-Goat anti-rabbit secondary antibody (1:400) for 30 min, and finally covered with 1.5 μg/ml DAPI (Beyotime). Images were collected and analyzed using a confocal microscope (Nikon A1R).
5
Immunohistochemistry of Vg in C. lividipennis Ovary
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The C. lividipennis adult ovaries were removed at 7 DAE, washed 3 times in cold PBS (pH = 7.2, 10 μmol/mL), for 2 h in 4% paraformaldehyde, and washed 3 times in PBS for 5 min. The ovaries were then washed 3 times in PBS containing Triton X-100 (PBST), blocked in PBST containing 5% goat serum, and incubated with anti-Vg (1:500) for 2 h, as mentioned previously [65 (link)]. After 5 min washes with PBS once, Alexa Fluor 488-labeled goat anti-rabbit secondary antibody (1:500) (Beyotime, Shanghai, China) was applied to PBST containing 2% goat serum and 3% bovine serum albumin. The nuclei were then counterstained in PBST for 10 min with 100 nM 4′,6-diamidino-2-phenylindole following incubation at room temperature for 1 h under low light (DAPI; Beyotime). Samples were put on slides and washed in PBS 3 times, 5 min per wash. The fluorescence photos were captured using a Carl Zeiss LSM 780 confocal microscope (Carl Zeiss MicroImaging, Shanghai, China) [20 (link)].
6
Fluorescent in situ Hybridization (FISH) Protocol
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The FISH assay was performed as previously described [24 (link)]. The Cy3-labeled JPX probes used in our study were designed and synthesized by GenePharma (Shanghai, China). Briefly, the prepared cells were fixed with 4% paraformaldehyde containing 0.5% Triton X-100 for 20 min. The cells were incubated with probes at 37 °C overnight. The cell nuclei were stained with DAPI (Beyotime). The staining results were observed using Zeiss confocal laser microscopy (Zeiss 510, Jena, Germany). For tissue RNA FISH, fresh frozen sections were fixed in 4% paraformaldehyde, followed by incubation with RNase A at 37 °C for 45 min. Slides were prehybridized with probe overnight at 37 °C. After hybridization, sections were stained with DAPI (Beyotime). Slides were visualized using Zeiss confocal laser microscopy (Zeiss 510, Jena, Germany).
7
Immunohistochemical and Apoptosis Evaluation
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Tissues were fixed in 4% formaldehyde and embedded in paraffin. The following primary antibodies were used for IHC staining at the indicated dilution: PLK1 (1:50, #4513, CST), p-p53 (1:200, ab76242, Abcam), p-Chk2 T68 (1:200, #2197, CST), Ki-67 (1:600, GB111499, Servicebio). The smears were scanned using Panoramic MIDI (3DHISTECH) and scored by two pathologists following the double-blind principle.
IF staining was performed as described previously.76 (link) The primary antibodies were against FOXO3a (1:400; #12829, CST; 66,428-1, Proteintech) and PLK1 (#4513, CST). Alexa Fluor 555-labeled donkey anti-rabbit IgG and FITC-labeled goat anti-mouse IgG (Beyotime) were used. Cell nuclei were stained with DAPI (Beyotime).
Fragmented DNA in situ labeling was performed with the One Step TUNEL Apoptosis Assay Kit (Beyotime). The DNA fragments were labeled with fluorescein-dideoxy-UTP by the TUNEL and the nuclei were counterstained with DAPI (Beyotime). The percentage of TUNEL-positive cells (apoptotic cells) was evaluated.
IF staining was performed as described previously.76 (link) The primary antibodies were against FOXO3a (1:400; #12829, CST; 66,428-1, Proteintech) and PLK1 (#4513, CST). Alexa Fluor 555-labeled donkey anti-rabbit IgG and FITC-labeled goat anti-mouse IgG (Beyotime) were used. Cell nuclei were stained with DAPI (Beyotime).
Fragmented DNA in situ labeling was performed with the One Step TUNEL Apoptosis Assay Kit (Beyotime). The DNA fragments were labeled with fluorescein-dideoxy-UTP by the TUNEL and the nuclei were counterstained with DAPI (Beyotime). The percentage of TUNEL-positive cells (apoptotic cells) was evaluated.
8
Exosome Uptake and Interaction Visualization
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Exosomes were stained with PKH-26 (2 µM, Sigma, St. Louis, MO, USA) for 30 min and washed twice in PBS to remove excessive PKH-26 dye. Subsequently, THP-1 cells were co-cultured with PKH-26-labeled exosomes or an equal volume of the PKH26-PBS control for 24 h and stained with DAPI (1 µg/mL, Beyotime, Shanghai, China) prior to examination under a fluorescence microscope. For co-staining of circRHCG and exosomes, Cy3-labeled circRHCG was transfected into MDA-MB-468 and MDA-MB-231, and exosomes were extracted from culture supernatants and labeled with PKH67 (Sigma) at 2 µM for 30 min. THP-1 cells were incubated with exosomes for 24 h and stained with DAPI (1 µg/mL, Beyotime) prior to examination under a fluorescence microscope.
9
Quantitative Apoptosis Assessment by Flow Cytometry
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Apoptosis was measured by the Annexin V‐FITC apoptosis detection kit (Beyotime Biotechnology) and DAPI nuclear staining. In brief, the cells were collected after each treatment, washed with PBS, and incubated with trypsin. Cell suspensions were collected and centrifuged, the supernatant was discarded, the pellets were washed with PBS, and the final concentrations were adjusted to 1 × 106–1 × 107 cells/ml. Cells were resuspended in 200 μl of 1× binding buffer (5 μl Annexin V‐FITC, 10 μl PI), incubated on ice for 15 min in the dark, and then analyzed by flow cytometry (FACSVantage SE; BD Biosciences, Carlsbad, CA). At least 10,000 event cells were counted and gated in four categories: live (Annexin‐V–/PI–), early apoptotic (Annexin‐V+PI−), late apoptotic (Annexin‐V+PI+), and necrotic (Annexin‐V–/PI+) cells. In addition, partial cells were fixed with 4% paraformaldehyde for 30 min after each treatment and stained with DAPI for 5 min in dark. After DAPI (Beyotime Biotechnology) nuclear staining, the cells were monitored under a fluorescence microscope (Leica Microsystems) for nuclear change. All of the experiments were repeated at least three times on independent samples.
10
Cellular Uptake of Fluorescent Polymer Beads
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CLSM was used to evaluate the uptake of PBs by cancer cells. To obtain PBs-FITC, PBs was stirred with fluorescein isothiocyanate (FITC, 5 mg, Sigma-Aldrich, Shanghai, China) at room temperature overnight in dark. The final product was washed with ethanol for several times to acquire PBs-FITC. 4T1 cells were seeded into the CLSM-specific dishes (35 mm × 10 mm, Corning Inc, New York, USA) at a density of 1 × 105, and incubated for 24 h. The culture media was replaced by PBs-FITC (1 mL, 100 μg mL−1, dispersed into DMEM containing 10% FBS), which were then cultured for 0, 1, 2, and 4 h, respectively. The cells were washed with PBS and DAPI (100 μL, Beyotime Biotechnology) diluted with methanol (DAPI: methanol = 1:10) was added to stain cell nuclei for 15 min. The cells were washed and imaged by CLSM.
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