This assay was performed using the CF488A-TUNEL Apoptosis Assay Kit (Biotium, #30063). The FFPE sections were deparaffinized using xylene and rehydrated in graded ethanol. After washing with PBS, sections were permeabilized with 20 mg/mL proteinase K in PBS for 30 min at 37 °C. After incubating with 100 μL TUNEL Equilibration Buffer (Component 99965) for 5 min, sections were incubated with 50 μL of TUNEL reaction mix for 2 h at 37 °C and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted using vibrance antifade mounting medium (Vector Laboratories, H-1700). The TUNEL-positive cells were determined by ImageJ.
Vibrance antifade mounting medium
Manufactured by Vector Laboratories
Vibrance antifade mounting medium is a laboratory product designed to preserve and protect fluorescent signals in mounted samples. It helps maintain the brightness and stability of fluorescent dyes used in microscopy and other applications.
Automatically generated - may contain errors
Lab products found in correlation
Sp8 spectral confocal microscope, by Leica (1 mentions)
Round bottom ultra low attachment 96 well plate, by Corning (1 mentions)
Carbo free blocking solution, by Vector Laboratories (1 mentions)
Mildform 10n, by Fujifilm (1 mentions)
Hybrid cell count application, by Keyence (1 mentions)
Tcs sp8x inverted confocal microscope, by Leica (1 mentions)
4 protocols using vibrance antifade mounting medium
1
Apoptosis Quantification in FFPE Tissue
2
Visualization of CDN Uptake in Tumor Spheroids
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Tumour spheroids were formed by seeding 10,000 MC38 cells per well in a 96 well round-bottom ultra-low attachment plate (Corning) and used after 5 d of growth. Tumoroids were incubated in complete cell media (DMEM with 10% FBS) with sulfo-Cy5-labelled LND-CDN or liposome-CDN at a concentration of 5.0 μM CDN and 1.0 μM dye for 24 h. Following incubation, tumoroids were washed twice with complete media, followed by a PBS buffer wash, and then fixed using 4% paraformaldehyde in PBS for 20 min at 4 °C. Glass slides were prepared for mounting samples by adding two layers of double-sided tape strips (∼5 mm wide) along the edges of the slide to prevent flattening of the tumoroids when the coverslip was added. Tumoroids were washed with PBS and then transferred to glass slides, and excess PBS was blotted away, Vectashield vibrance antifade mounting medium was added, and a coverslip was added. Samples were imaged on a Leica SP8 spectral confocal microscope and analysed using ImageJ.
3
Quantification of Vascular Endothelial Cadherin in High Endothelial Venules
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Tissues were fixed in Mildform 10 N (Fujifilm Wako, Osaka, Japan) for 16–20 h at 4 °C and then embedded in paraffin. Three micrometer-thick sections were stained with HE after deparaffinization. For immunohistochemical analysis, 3 µm-thick sections were blocked with 5% BSA/phosphate-buffered saline (PBS; 137 mM NaCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl) or a Carbo-free blocking solution (for lectin staining, Vector laboratories, Newark, CA) for 1 h at 23–26 °C after deparaffinization. Next, these sections were incubated with primary antibodies or lectins for 16–20 h at 4 °C. Samples were visualized using Alexa Flour fluorescence-conjugated secondary antibodies (Thermo Fisher Scientific). Nuclei were counterstained with Vibrance Antifade Mounting Medium containing DAPI (Vector laboratories). All sections were observed using a BIOREVO BZ-X800 microscope (Keyence, Osaka, Japan).
VE-cadherin fluorescence intensity was quantified using the hybrid cell count application (Keyence). After the HEV area with positive PNAd signals was manually encircled, the fluorescence intensity of VE-cadherin was automatically quantified in the PNAd-positive area.
VE-cadherin fluorescence intensity was quantified using the hybrid cell count application (Keyence). After the HEV area with positive PNAd signals was manually encircled, the fluorescence intensity of VE-cadherin was automatically quantified in the PNAd-positive area.
4
Quantification of Intracellular Lipid Uptake
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About 60,000 J774A.1 cells in 300 µL culture medium were seeded into 8-well cell culture slides. After a 24 h preincubation, the cells were treated for 1 h with 100 µL of culture medium containing rHDL or Eve-rHDL, which had been labeled with DiI. Concentrations of 0.15 mM (lipid component amount) were used, which translated to 1.5 µM DiI and 18 µM Eve for Eve-rHDL. Concentrations of 50 nM LysoTracker™ Deep Red were used to stain acidic cell organelles. Multiple controls for each individual dye and the respective dye combinations were established to assess cellular autofluorescence and spectral overlap.
The cells were washed and incubated for 15 min with a 3% Pierce™ formaldehyde solution in PBS at room temperature in the dark. The samples were mounted with Vectashield® Vibrance™ Antifade mounting medium w/ DAPI and stored overnight at room temperature in the dark. Imaging was performed on a Leica TCS SP8 X inverted confocal microscope with 63×/1.40 oil objective magnification. DAPI fluorescence was recorded with a PMT detector at 419 to 484 nm, DiI fluorescence was recorded with a HyD SMD detector at 561 to 606 nm, and the lysotracker fluorescence was recorded with a HyD SMD detector at 657 to 764 nm. For sample excitation, lasers at 405, 553, and 647 nm were used. The illumination settings were kept fixed during the analysis.
The cells were washed and incubated for 15 min with a 3% Pierce™ formaldehyde solution in PBS at room temperature in the dark. The samples were mounted with Vectashield® Vibrance™ Antifade mounting medium w/ DAPI and stored overnight at room temperature in the dark. Imaging was performed on a Leica TCS SP8 X inverted confocal microscope with 63×/1.40 oil objective magnification. DAPI fluorescence was recorded with a PMT detector at 419 to 484 nm, DiI fluorescence was recorded with a HyD SMD detector at 561 to 606 nm, and the lysotracker fluorescence was recorded with a HyD SMD detector at 657 to 764 nm. For sample excitation, lasers at 405, 553, and 647 nm were used. The illumination settings were kept fixed during the analysis.
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