Confocal fluorescence microscopy analysis was conducted on a Leica SP5 inverted confocal laser scanning microscope. The microscope was equipped with a 63x oil-immersion objective and 405, 488, 453 and 633 nm excitation lasers. Gain and offset settings were optimized for each fluorescent channel within an experiment. Images were recorded using the sequential scanning mode to prevent fluorescence channel crosstalk/ bleed-through. Images were scanned at 100 Hz with a line average of three to reduce noise.
Sp5 inverted confocal laser scanning microscope
Manufactured by Leica
Sourced in Germany
The SP5 inverted confocal laser scanning microscope is a high-performance imaging system designed for advanced biological and materials research. It features a modular design, allowing for the integration of various laser sources, detectors, and accessories to meet the specific needs of the user. The SP5 provides high-resolution, real-time imaging capabilities, enabling the visualization and analysis of complex samples at the cellular and subcellular levels.
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Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Ab8898, by Abcam (2 mentions)
Anti h3k27me3 c15410195, by Diagenode (2 mentions)
Anti gfp 11814460001, by Merck Group (2 mentions)
Alexa fluor 488 anti mouse and 568 anti rabbit iggs, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Dmem hg, by Thermo Fisher Scientific (2 mentions)
Dylight fluors, by Jackson ImmunoResearch (1 mentions)
Application suite x 3, by Leica (1 mentions)
Draq5 nuclear stain, by Abcam (1 mentions)
Tunel enzyme, by Roche (1 mentions)
Tunel label, by Roche (1 mentions)
Rabbit anti smad2 3, by Cell Signaling Technology (1 mentions)
Sc 136083, by Santa Cruz Biotechnology (1 mentions)
Cas block, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Application suite advanced fluorescence lite software, by Leica (1 mentions)
Las af software, by Leica (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
18 protocols using sp5 inverted confocal laser scanning microscope
1
Confocal Fluorescence Microscopy Imaging
2
Immunofluorescence Staining of Histone Modifications
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Cells were seeded and grown as described above, fixed in 4% formaldehyde in PBS for 10 min at room temperature, washed three times in PBS, permeabilized for 5 min at room temperature in PBS supplemented with 0.1% Triton X-100 and 0.25% BSA (Sigma-Aldrich), and washed twice in PBS. Corresponding primary antibodies anti-H3K9me3: ab8898 (abcam), anti-H3K27me3: C15410195 (diagenode), anti-GFP: 11814460001 (Millipore) and secondary antibodies (Alexa Fluor 488 anti-mouse and 568 anti-rabbit IgGs from ThermoFisher) were diluted in PBS containing 2% FBS and 0.02% BSA. Primary antibody incubations were performed overnight at 4°C. Secondary antibody incubations were performed for 1h at room temperature. Following antibody incubations, cells were washed once with PBS and incubated for 10 min with PBS containing 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI, 0.5 μg/ml) for 10 min at room temperature to stain DNA. Randomly selected cells were imaged with sequential acquisition settings on a Leica SP5 inverted confocal laser scanning microscope.
3
Confocal Fluorescence Microscopy Optimization
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Confocal fluorescence microscopy analysis was conducted on a Leica SP5 inverted confocal laser scanning microscope. The microscope was equipped with a 63x oil-immersion objective and the 405 and 488 nm lasers were used. Gain and offset settings were optimised for each fluorescent channel within an experiment. Images were recorded using the sequential scanning mode to prevent fluorescence channel crosstalk/bleed-through. Images were scanned at 100 Hz with a line average of three to reduce noise.
4
Localized UV Irradiation of Melanoma Cells
5
Visualizing Staphylococcus-Neutrophil Interactions
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A Leica SP5 inverted confocal laser scanning microscope was utilized for all imaging. GFP-tagged bacteria and PMNs were excited with the 488 nm and 561 nm laser lines, respectively. A LiveCell (Pathology Devices, CA, USA) environmental chamber system was utilized to maintain 5% CO2, 20% O2, 50% humidity, and 37°C for sample incubation during imaging. Image stacks 12–20 μm in size with 1-μm z-slices were taken sequentially at 2–3 min intervals over a 4-h time course using a Leica 20x/0.7 NA dry objective lens. At least two fields of view from each chamber of the dish were generally imaged per experiment. FOVs with large numbers of bacterial objects were selected for imaging to visualize the highest number of S. aureus—PMN interactions.
6
Immunofluorescence Staining of Histone Modifications
7
Quantifying Nuclear Lamina RPA Foci
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Confocal images were acquired on an automated Leica SP5 inverted confocal laser scanning microscope equipped with diode, argon, and helium neon lasers (405 nm, 488 nm, 561 nm, 633 nm) using an HCX PL APO Leica 63x immersion oil objective (NA 1.4). RPA foci co-localization with the nuclear lamina was quantified from 3D z stacks.
8
Confocal Imaging with Leica SP5 Microscope
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Confocal images were acquired on an automated Leica SP5 inverted confocal laser scanning microscope equipped with Argon lasers for 453 nm, 476 nm, 488 nm, 496 nm and 514 nm, and a diode laser for 561 nm. Confocal images were acquired with an HCX PL APO Leica 63x oil immersion objective with PMT detectors using Leica Application Suite X 3.6.0.20104 and Leica LAS AF Version 2.7.3.9723.
9
Silencing Fshr via Accell siRNA in Preantral Follicles
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Size-matched preantral follicles were mechanically isolated from mice aged 15 to 16 days as described above. Single follicles were cultured in individual wells in 96 well plates with 1 µm Accell small interfering RNA (siRNA) designed to target exon 8 of the mouse Fshr transcript (GCGAUAACAAUAAUUUGGA; A-062874-17; ThermoScientific). Control follicles were exposed to Accell Nontargeting siRNA (D-001910-01; ThermoScientific) or Accell Red Nontargeting siRNA, containing a DY-547 label (D-001960-01; ThermoScientific). After 24 hours, 10 ng/mL rhFSH was added to all groups, with the exception of an additional control group. Follicles were maintained in culture for another 96 hours and were photographed daily using light microscopy for assessment of growth as described above. Fshr messenger RNA (mRNA) expression was reduced by 50% relative to nontargeting controls (data not shown). Some of the follicles exposed to labeled siRNA were incubated in 5 µm DRAQ5 nuclear stain (Abcam; Cambridge, UK) for 10 min before imaging in chamber slides using a Leica inverted SP5 confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany) to visualize penetration of siRNA into the follicle.
10
Apoptotic Cell Detection in Ovarian Tissue
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Apoptotic cells in ovarian tissue sections were identified with an in situ Cell Death Detection Kit, POD (Roche, Germany) according to the manufacturer's instructions. Paraffin sections of the ovaries were dewaxed, rehydrated, digested with proteinase K (10 µM) for 5 min, incubated in TUNEL enzyme (10% v/v; Roche) and TUNEL label (90% v/v; Roche) for 60 min at 37°C, and mounted in ProLong Gold medium containing 4',6-diamidino-2-phenylindole (DAPI). Negative control sections were incubated with a TUNEL reaction mixture without enzyme (terminal deoxynucleotidyl transferase, TdT). Finally, the sections were observed and digital images were recorded using a Leica inverted SP5 confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany).
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