Ishikawa cells were grown on 14 mm glass coverslips at a density of 2 × 10 5 cells per well in a 24-well plate. 10 ng/mL TNF-α and 25 µg/mL ETA were used to treat the cell line. After 48 h of treatment, the cells were analyzed with the TUNEL BrightGreen Apoptosis Detection Kit (Vazyme, Inc., Nanjing, China), followed by mounting with antifade mounting medium with DAPI (Beyotime, Inc.) The analysis was conducted using a confocal laser scanning microscope (Olympus FV1000). The DNase (Vazyme, Inc., Nanjing, China) treated control group cells served as positive control. Each test had two replicates (n = 3).
Antifade mounting medium with dapi
Manufactured by Beyotime
Sourced in China
Antifade Mounting Medium with DAPI is a solution designed for use in fluorescence microscopy. It serves to preserve the fluorescence of labeled samples and stain cell nuclei with DAPI (4',6-diamidino-2-phenylindole), a common DNA-binding dye.
Automatically generated - may contain errors
Lab products found in correlation
Fv1000, by Olympus (6 mentions)
Immunol staining blocking buffer, by Beyotime (4 mentions)
One step tunel apoptosis assay kit, by Beyotime (4 mentions)
Paraformaldehyde (pfa), by Solarbio (4 mentions)
Fv3000, by Olympus (4 mentions)
Dnase, by Vazyme (3 mentions)
Tunel brightgreen apoptosis detection kit, by Vazyme (3 mentions)
N storm a1, by Nikon (3 mentions)
Tcs sp8, by Leica (3 mentions)
Triton x 100, by Beyotime (3 mentions)
Tunel apoptosis assay kit, by Beyotime (3 mentions)
Alexa fluor 594conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg h l, by Thermo Fisher Scientific (2 mentions)
Primary antibodies for tnf α, by Proteintech (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Tc5 sp5 confocal microscope, by Leica (2 mentions)
Coralite594 conjugated goat anti rabbit igg antibody, by Proteintech (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Bovine serum albumin (bsa), by Beyotime (2 mentions)
Dnase free proteinase k, by Beyotime (2 mentions)
86 protocols using antifade mounting medium with dapi
1
Apoptosis Analysis of Ishikawa Cells
2
Immunofluorescence Staining of Embryos
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The embryo was xed with 4% PFA for 15 min at room temperature and 0.3% Triton X-100 in PBS was added for 20 min to permeabilize the cell membrane. Then, the embryo was incubated with primary antibodies for TNF-α (1:100, Proteintech Inc., Rosemont, IL, USA) and CDX2 (1:100, Abcam, Inc., Cambridge, MA, USA) at 4 °C overnight. Subsequently, the embryo was incubated with Alexa Fluor 594conjugated goat anti-mouse IgG (H + L), (1:1000, Invitrogen, Inc., Carlsbad, CA, USA) and AlexaFluor 488conjugated goat anti-rabbit IgG (H + L) (1:1000, Invitrogen, Inc.) for 60 min at room temperature in the dark, followed by mounting with antifade mounting medium with DAPI (Beyotime, Inc., Shanghai, China).
The analysis was conducted using a confocal laser scanning microscope (Olympus FV1000, Olympus Corporation, Waltham, MA, USA) (Nikon N-STORM & A1, Nikon Corporation, Tokyo, Japan). The six embryos used for staining originated from the same volunteer.
The analysis was conducted using a confocal laser scanning microscope (Olympus FV1000, Olympus Corporation, Waltham, MA, USA) (Nikon N-STORM & A1, Nikon Corporation, Tokyo, Japan). The six embryos used for staining originated from the same volunteer.
3
Colocalization of CD71 and HFn-FITC in C666-1 Cells
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Fluorescence microscopy analysis
was used to show the colocalization of CD71 and HFn-FITC within CD71-positive
C666-1 cells. Upon reaching 90% confluence, C666-1 cells were detached,
washed, and then fixed with 4% paraformaldehyde for 15 min at 37 °C.
After washing and suspending in phosphate-buffered saline (PBS), a
20 μL aliquot of the cell suspension was placed onto a slide,
allowed to sit for 20 min at room temperature, and then dried in an
oven at 37 °C. The cell smears were permeabilized with 0.1% Triton
X-100 for 15 min and blocked with 3% bovine serum albumin (BSA) for
1 h at room temperature. The cells were then incubated with the following
reagents: 5 μg/mL of mouse IgG1 kappa isotype control (Invitrogen,
USA), 5 μg/mL of CD71 mouse monoclonal antibody (Invitrogen,
USA), and 10 μg/mL of HFn-FITC in 0.1% BSA at 4 °C overnight.
Subsequently, the cells were labeled with Alexa Fluor 488 and Alexa
Fluor 555 secondary goat antimouse antibody (Bioss, China) at a dilution
of 1:100 for 1 h at room temperature. After rinsing with PBS containing
0.05% tween 20 (Solarbio, China), an antifade mounting medium with
DAPI (Beyotime Biotechnology, China) was applied to stain the cell
nuclei for 10 min. Finally, the cell smears were examined using a
confocal laser scanning microscope with 405, 488, and 543 nm wavelengths
of laser (Zeiss LSM880, Carl Zeiss AG, Oberkochen, Germany).
was used to show the colocalization of CD71 and HFn-FITC within CD71-positive
C666-1 cells. Upon reaching 90% confluence, C666-1 cells were detached,
washed, and then fixed with 4% paraformaldehyde for 15 min at 37 °C.
After washing and suspending in phosphate-buffered saline (PBS), a
20 μL aliquot of the cell suspension was placed onto a slide,
allowed to sit for 20 min at room temperature, and then dried in an
oven at 37 °C. The cell smears were permeabilized with 0.1% Triton
X-100 for 15 min and blocked with 3% bovine serum albumin (BSA) for
1 h at room temperature. The cells were then incubated with the following
reagents: 5 μg/mL of mouse IgG1 kappa isotype control (Invitrogen,
USA), 5 μg/mL of CD71 mouse monoclonal antibody (Invitrogen,
USA), and 10 μg/mL of HFn-FITC in 0.1% BSA at 4 °C overnight.
Subsequently, the cells were labeled with Alexa Fluor 488 and Alexa
Fluor 555 secondary goat antimouse antibody (Bioss, China) at a dilution
of 1:100 for 1 h at room temperature. After rinsing with PBS containing
0.05% tween 20 (Solarbio, China), an antifade mounting medium with
DAPI (Beyotime Biotechnology, China) was applied to stain the cell
nuclei for 10 min. Finally, the cell smears were examined using a
confocal laser scanning microscope with 405, 488, and 543 nm wavelengths
of laser (Zeiss LSM880, Carl Zeiss AG, Oberkochen, Germany).
4
Immunofluorescence Staining of Ana-1 Cells
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Following different stimulations, we collected Ana-1 cells and adjusted the concentration to 105–2 × 105/mL. Then, we added 100 µL of cell suspension to a smear and slide centrifuge and centrifuged at 400g for 3 min to obtain cell smears. Consequently, we fixed the cells with 4% formaldehyde for 10 min and gently washed them thrice with PBS. Next, the smear was covered with 0.3% Triton X-100 drops for 10 min and blocked with Immunol Staining Blocking Buffer (Beyotime, P0102) for 15 min, followed by overnight incubation with primary antibody at 4 °C. The following day, the cells were washed thrice with PBS and incubated with fluorescent secondary antibody for 1 h in the dark at room temperature. The cells were washed thrice with PBS, and the nuclei were visualized by adding an Antifade Mounting Medium with DAPI (Beyotime, P0131).
5
Multifunctional Dendritic Nanosensor
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Carboxyl PEG2k‐N3 and Methoxyl PEG2k carboxyl were purchased from Ponsure Biological. Sodium ascorbate, Tri[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl) methyl] amine (TBTA), and CuI were purchased from Accela ChemBio Co., Ltd. (Shanghai, China). N‐(3‐Dimethylaminopropyl)‐N’‐ethylcarbodiimide hydrochloride (EDC), 1‐Hydroxybenzotriazole (HOBt), N‐Hydroxysuccinimide (NHS), 4‐Dimethylaminopyridine (DMAP), and N,N‐Diisopropylethylamine (DIPEA) were purchased from Energy Chemical (Shanghai, China). PAMAM 5.0 was purchased from Weihai CY Dendrimer Technology Co., Ltd. (Shandong, China). 3‐Hydroxy‐4‐nitrobenzaldehyde was purchased from Shanghai Haohong Biomedical Technology Co., Ltd. (Shanghai, China). Propargyl bromide was purchased from Macklin Biochemical Co., Ltd. 4‐Nitrophenyl chloroformate and DPBF from Sigma–Aldrich. Antifade Mounting Medium with DAPI was purchased from Beyotime Biotech. Inc. (Shanghai, China). Resiquimod (R848) was purchased from Jiangsu Aikon (Jiangsu, China). Detailed information on the antibodies such as fluorescent antibody type, manufacturer, clone, and catalog number used in this study was provided in Tables S1 –S3 (Supporting Information).
6
Immunofluorescence Assay for γ-H2AX Foci
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Immunofluorescence assay was used to detect the number of γ-H2AX foci. Cells were seeded in 24-well plates. After washing with PBS, cells were fixed in 3% paraformaldehyde and permeabilized in 0.1% Triton X-100 in PBS. The cells were then stained with γ-H2AX primary antibody (Cell Signaling Technology; 1:200) and thereafter the secondary antibody (fluorescein goat anti-rabbit IgG, Invitrogen; 1:1000). Cells were stained with a DAPI (Antifade Mounting Medium with DAPI, Beyotime). The images were captured using an Olympus BX60 fluorescent microscope (Olympus America Inc).
7
Immunofluorescence Detection of DNA Damage
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Cells were fixed with 4% formaldehyde in PBS after drug treatment, blocked using 5% BSA, and permeabilized with 0.2% Triton X‐100. The primary antibodies were diluted in 1% BSA and incubated at 4°C overnight. Then, secondary antibodies were added to the samples and incubated at room temperature for 1 hour. Antibodies against RAD51 (Proteintech, 14961‐1‐AP, 1:200) and γH2AX (Cell Signaling Technology, #2577,1:200) were used as the primary antibodies. Antifade Mounting Medium with DAPI was from Beyotime (China). Fluorescent secondary antibodies were used and images were captured with a fluorescence microscope (Olympus, Japan).
8
Histological Analysis of Mouse Brain Tissue
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The brain tissues were prepared as described above. Isolated mice’s brains were fixed in 4% paraformaldehyde overnight, and then embedded in paraffin. Paraffin blocks were cut into 5 μm-thick sections, which were de-paraffinized and rehydrated after mounting on glass slides. Hematoxylin and eosin (H&E) staining was performed using the H and E dye solution set (Servicebio, Wuhan, China, G1003). Sections were examined using the Digital Slide Scanner (Winmedic, Shandong, China). Immunostaining was performed as following: samples were permeabilized in 0.2% Triton X-100 in TBS for 5 min and were then washed three times in TBS. After blocking in 5% horse serum in TBS at room temperature for 1 h, slides were incubated with anti-GFAP (1:1000, Servicebio, GB12096) overnight at 4 °C, placed in a wet box. GFAP immunome activity was detected with Cy3 conjugated Goat Anti-mouse IgG (1:4000; Servicebio, GB27301). Stained samples were mounted with Antifade Mounting Medium with DAPI (Beyotime, Nantong, China, P0131) and visualized by confocal microscopy (Olympus Fluoview FV1000).
9
Glioma Cell Immunofluorescence Imaging
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U251 and A172 cells were seeded on 20 × 20 mm glass-bottom round dishes (Corning, NY, USA) and cultured overnight to 50% confluence. Cells were fixed with 4% paraformaldehyde for 20 min and subsequently permeabilized with 0.1% Triton X-100 before being blocked by 5% bovine serum albumin for 30 min at room temperature. The primary antibodies were added to glioma cells and incubated at 4 °C overnight. After washing with PBS, cells were incubated with secondary antibodies for 1 h away from light. Antifade Mounting Medium with DAPI (Beyotime Biotechnology, Shanghai, China) was utilized to stain nuclei for 10 min, and then a Leica TC5 SP5 confocal microscope was adopted to visualize and image the cells. Antibodies are listed in Supplementary Table S4 .
10
Actin Visualization and Quantification
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Actin-Tracker Green (Beyotime, Shanghai, China, C1033) was diluted with Immunol Fluorescence Staining Secondary Antibody Dilution Buffer (Beyotime, Shanghai, China, P0108) to 1:100. Approximately 5 × 10 3 cells were seeded in glass-bottom cell culture dishes (NEST, Wuxi, China, 801001), then xed with 4% paraformaldehyde for 30 min and incubated in diluted Actin-Tracker Green for 1 h at 25℃ in the dark. After washing with PBS three times, the dish was incubated with Antifade Mounting Medium with DAPI (Beyotime, Shanghai, China, P0131). Actin viability with different drug treatments was determined using uorescence microscopy.
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