Cells grown as monolayers on coverslips were first fixed in 4% paraformaldehyde for 20 min and then permeabilized in 0.5% Triton X-100 for 1 min or treated with the reverse protocol. After washes with PBS, the cells were blocked in 5% FBS for 1 h and incubated with the indicated antibodies followed by DAPI staining. Images were acquired using an Olympus BX51 fluorescence microscope and the Leica TCS-SP2 Spectral Confocal System.
Bx51 fluorescence microscope
Manufactured by Leica
The Leica BX51 is a fluorescence microscope designed for scientific research and analysis. It features a high-performance optical system, allowing for clear and detailed observation of fluorescently labeled samples. The BX51 is capable of providing high-quality images and data for a variety of applications, such as cell biology, molecular biology, and material science.
Automatically generated - may contain errors
3 protocols using bx51 fluorescence microscope
1
Immunofluorescence Staining of Cells
2
Fluorescence-based TUNEL Assay for Apoptosis
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An AAT Bioquest TUNEL Apoptosis Assay kit
(Catalog #22844) was used for the detection of DNA fragmentation.
The kit works by incorporating the fluorescence dye TF3-dUTP at the
3′-OH ends of apoptotic DNA fragments. After the expression
of the protein of interest, OD600 was measured, and cells
were counted, and 30 000 cells were pipetted on to wells of a 96-well
microtiter plate (Corning, 04815027) in triplicate. The plate was
then centrifuged for 2 min at 800 rpm. Supernatants were removed,
and 100 μL of 4% formaldehyde fixative buffer was added to each
well and incubated for 30 min at room temperature. The fixative buffer
was removed and cells were washed twice with PBS. Component A (6 μL,
100× TF3-dUTP) was added to 600 μL of component B (reaction
buffer). The abovementioned mixture (50 μL) was added to wells
of a 96-well microtiter plate and incubated for 120 min. Cells were
then washed and fluorescence was monitored from the bottom with the
help of a Bio-Tek Synergy HT plate reader at excitation/emission 550/590
nm. An Olympus BX-51 fluorescence microscope with a Leica digital
imaging camera was used to take the microscopic images.
(Catalog #22844) was used for the detection of DNA fragmentation.
The kit works by incorporating the fluorescence dye TF3-dUTP at the
3′-OH ends of apoptotic DNA fragments. After the expression
of the protein of interest, OD600 was measured, and cells
were counted, and 30 000 cells were pipetted on to wells of a 96-well
microtiter plate (Corning, 04815027) in triplicate. The plate was
then centrifuged for 2 min at 800 rpm. Supernatants were removed,
and 100 μL of 4% formaldehyde fixative buffer was added to each
well and incubated for 30 min at room temperature. The fixative buffer
was removed and cells were washed twice with PBS. Component A (6 μL,
100× TF3-dUTP) was added to 600 μL of component B (reaction
buffer). The abovementioned mixture (50 μL) was added to wells
of a 96-well microtiter plate and incubated for 120 min. Cells were
then washed and fluorescence was monitored from the bottom with the
help of a Bio-Tek Synergy HT plate reader at excitation/emission 550/590
nm. An Olympus BX-51 fluorescence microscope with a Leica digital
imaging camera was used to take the microscopic images.
3
Autophagy Evaluation in Parasites
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Parasites before and after incubation with 19 μM R72 for 24 h were fixed in formaldehyde, washed in PBS, adhered in poly-L-lysine-coated coverslips and stained with 0.05 mM monodansylcadaverine (MDC) or 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) for detections localization of autophagic and nuclear compartments, respectively. Percentage cells displaying punctate staining was determine by examination of over 500 cells per experiment. Statistical analysis was performed using chi-square with Yates correction (1 degree of freedom), with P < 0.0001 (two-tailed). Samples were analyzed in an Olympus BX51fluorescence microscope or confocal microscope Leica SP8.
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