For histology, the left lungs were removed and immediately fixed in 4% paraformaldehyde (PFA) for 24 h. The samples were then dehydrated, embedded in paraffin, sectioned into 3- to 4-µm slices, and stained with periodic acid–Schiff reagent (PAS) (Carl Roth GmbH, Karlsruhe, Germany). In situ TUNEL assays were performed with an in situ Cell Death Detection kit, TMR red (Roche Diagnostics, Mannheim, Germany). All of the steps were performed according to the supplier’s instructions. Briefly, the paraffin-embedded sections were dewaxed, rehydrated, permeabilized in 0.1 M Na-citrate buffer pH 6.0 (microwave irradiation, 5 min, 350 W), and washed twice with PBS. The sections were then incubated for 60 min at 37 °C in a humidified chamber with a labeling solution containing terminal deoxynucleotidyl transferase. They were then washed twice with PBS and embedded with Mowiol (Carl Roth GmbH). The images were analyzed with Leica Confocal Software version 2.61 (Leica Microsystems, Mannheim, Germany). Tissue sections treated with DNAse I (1 mg/mL in Ca2+/Mg2+ buffer; Roche Diagnostics) were used as positive controls.
Confocal software version 2
Manufactured by Leica
Sourced in Germany
Leica Confocal Software version 2.61 is a software suite designed to operate and control Leica confocal microscopy systems. It provides the necessary tools and functionality to acquire, process, and analyze confocal imaging data.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp2 confocal microscope, by Leica (4 mentions)
Mowiol, by Carl Roth (1 mentions)
Dnase 1, by Roche (1 mentions)
Tmr red, by Roche (1 mentions)
Labtek 2 chambers, by Thermo Fisher Scientific (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Ibitreat chamber slides, by Ibidi (1 mentions)
Tcs sp2 aobs, by Leica (1 mentions)
Tetramethylrhodamine isothiocyanate tritc phalloidin, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Dapi stain solution, by Thermo Fisher Scientific (1 mentions)
Tcs sp2 a0bs confocal system, by Leica (1 mentions)
Vectashield mounting medium with 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions)
7 protocols using confocal software version 2
1
Histological Analysis of Lung Tissue
2
Visualizing Ras-Raf Signaling Dynamics
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CH7C17 cells (15 × 106) were allowed to settle in LabTek II chambers (Nalge Nunc International, Sigma-Aldrich Quimica SL, Madrid, Spain) and maintained at 37 °C in a 5% CO2 atmosphere in phenol red-free RPMI medium 1640 containing 25 mM HEPES and 2% FBS in an incubator coupled to a Leica TCS SP2 confocal microscope (Leica Microsistemas S.L.U.). T cells transiently co-transfected with YFP-RBD-Raf-1 and CFP-H-Ras were serum starved at 37 °C for 2 h before stimulation with DMSO, PGA1 (30 μM/15 min), or anti-human CD3ɛ T3b mAb (5 μg/ml). Time-lapse confocal images were collected with an HCX PL APO × 40 NA 1.32 oil-immersion objective lens (Leica), and fluorescence images were captured every 1 min. Six confocal Z-sections were necessary to capture the entire fluorescent signal at each time. The ratio of fluorescence intensity between the cellular regions of interest was calculated in a single Z plane, corresponding with the maximum fluorescence plane, by using Leica Confocal Software, version 2.61 (Leica Microsystems, Leica Microsistemas S.L.U.). At least 100 cells were analyzed for each sample. For fluorescence profile analysis, 8 μm cross-sections were drawn (white bar).
3
Imaging Cyanobacterial Cells and Vancomycin Staining
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Exponentially growing cells (5 μL) were mounted on 2% low-melting-point agarose pads for microscopy. To image cell auto-fluorescence and vancomycin staining (BODIPY™ FL vancomycin; final concentration 1 μg ml–1, 20 min 37°C), a TCS-SP2 Leica confocal microscope was used as described in Labella et al. (2017) (link) (running under Leica Confocal Software version 2.61, Leica Microsystems). The filter specificities were as follows: ex633 nm/em665–700 nm (for cyanobacterial auto-fluorescence analysis) and ex488 nm/em495–537 nm (for vancomycin).
To visualize DNA compaction, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) at 8.9 μg ml–1 for 10 min at 30°C. Micrographs were taken using a confocal laser scanning microscope (Zeiss model LSM800). The filter parameters were as follows: ex640 nm/em650+ nm (for cyanobacterial auto-fluorescence analysis) and ex405 nm/em410–470 nm (for DAPI).
To visualize DNA compaction, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) at 8.9 μg ml–1 for 10 min at 30°C. Micrographs were taken using a confocal laser scanning microscope (Zeiss model LSM800). The filter parameters were as follows: ex640 nm/em650+ nm (for cyanobacterial auto-fluorescence analysis) and ex405 nm/em410–470 nm (for DAPI).
4
Subcellular Actin Localization in Macrophages
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To analyze the subcellular localization of actin, different human macrophage subtypes were cultured in the presence of storage buffer (vehicle control) or 10 μg/ml recombinant human galectin-2 at day 7 on ibitreat chamber slides (Ibidi, Planegg/Martinsried, Germany) at a density of 1 x 106 cells/ml in DMEM complete medium. Macrophages were fixed in 4% PFA in HBSS (Invitrogen) at RT for 10 minutes, and permeabilized with 0.1% Triton X-100 (Merck) in PBS at RT for 5 minutes. Finally, the cells were stained with 0.4 μg/ml phalloidin-tetramethyl rhodamine iso-thiocyanate (TRITC; Sigma-Aldrich) at RT for one hour. Imaging was performed by a confocal laser scanning microscope (Leica TCS SP2 AOBS, Leica Microsystems B.V., Rijswijk, The Netherlands). A total of five randomly selected fields at 63 times original magnification were acquired with Leica confocal software version 2.61 (Leica Microsystems, Wetzlar, Germany).
5
Quantifying Motor Neuron Changes in ALS
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Single confocal planes of randomly chosen areas of ventral horn sections from three control donors and three ALS patients, stained as described above, were acquired by Leica TCS SP2 confocal microscope (Leica Microsystems, Heidelberg GmbH). Leica Confocal Software Version 2.61 was used to measure motor neuron size and to quantify SNAP25 and STX1A expression levels. Motor neuron cell bodies were drawn based on the β3 tubulin staining, and the surface quantified and expressed in µm2. The average signal value relative to SNAP25 and STX1A for each motor neuron was also calculated and normalized on the motor neuron surface. Results were expressed as means ± standard error. Statistical significance was evaluated using unpaired Student’s t test. Difference with p < 0.05 were considered statistically significant.
6
Visualizing CLL Cells Using Confocal Microscopy
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Total CLL cell population from patients (106 cells/mL) were incubated with PE-conjugated anti-CD5 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) and FITC-conjugated p1 peptide (10 μg/mL) and DAPI on ice for 20 min in the dark. After washing with Perm/WashTM Buffer solution (Becton Dickinson Italia S.p.A, Milan, Italy), cells were incubated with DAPI stain solution (Thermo Fisher, Waltham, MA, USA) at room temperature for 5 min in the dark. After extensive washing, cells were mounted under a cover slip, and visualized by confocal microscopy. Pictures were captured with a Leica TCS SP2 confocal microscope with a HCX PL APO 63.0×/1.40 oil UV objective (NA1.40) in glycerol and acquired with Leica Confocal Software Version 2.61 (Wetzlar, Germany). Image manipulation was performed with Adobe Photoshop CC 2015 software (San Jose, CA, USA)
7
Immunofluorescence Analysis of CFTR Expression
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The A549 cells cultured in collagen-coated cover slides were transfected with
pacAd5.huCFTR-ECL4HA plasmid for 12 hours and cultured in the presence or absence of
nicotine for additional 24 hours. The cells were then fixed with 4% paraformaldehyde in
phosphate-buffered saline (PBS) at room temperature for 15 minutes, washed in PBS for 3× 5
minutes, and permeabilized with 0.3% Triton X-100 for 10 minutes at room temperature.
Nonspecific antibody binding was blocked using 5% normal donkey serum in PBS for 1 hour at
room temperature, after which primary antibodies of mouse anti-CFTR and rabbit anti-HA
were applied at a 1:100 dilution in PBS and applied to probe proteins of interest by
incubating slides at 4°C overnight. The primary antibody binding was detected using the
Alexa Fluor 488-labeled donkey-anti-mouse immunoglobulin G (IgG) secondary antibody
(1:500) and Alexa Fluor 588-conjugated donkey antirabbit IgG (1:500; Jackson
ImmunoResearch Lab, West Grove, Pennsylvania). After extensively washing, the slides were
mounted for fluorescence in Vectashield Mounting Medium with 4′,6-diamidino-2-phenylindole
(DAPI) (Vector Lab, Burlingame, California). Images were acquired using a Leica TCS SP2
A0BS Confocal System and processed on Leica Confocal Software version 2.6.1 (Leica,
Wetzlar, Germany). Detailed information of antibodies used in this study is listed in
Table 1 .
pacAd5.huCFTR-ECL4HA plasmid for 12 hours and cultured in the presence or absence of
nicotine for additional 24 hours. The cells were then fixed with 4% paraformaldehyde in
phosphate-buffered saline (PBS) at room temperature for 15 minutes, washed in PBS for 3× 5
minutes, and permeabilized with 0.3% Triton X-100 for 10 minutes at room temperature.
Nonspecific antibody binding was blocked using 5% normal donkey serum in PBS for 1 hour at
room temperature, after which primary antibodies of mouse anti-CFTR and rabbit anti-HA
were applied at a 1:100 dilution in PBS and applied to probe proteins of interest by
incubating slides at 4°C overnight. The primary antibody binding was detected using the
Alexa Fluor 488-labeled donkey-anti-mouse immunoglobulin G (IgG) secondary antibody
(1:500) and Alexa Fluor 588-conjugated donkey antirabbit IgG (1:500; Jackson
ImmunoResearch Lab, West Grove, Pennsylvania). After extensively washing, the slides were
mounted for fluorescence in Vectashield Mounting Medium with 4′,6-diamidino-2-phenylindole
(DAPI) (Vector Lab, Burlingame, California). Images were acquired using a Leica TCS SP2
A0BS Confocal System and processed on Leica Confocal Software version 2.6.1 (Leica,
Wetzlar, Germany). Detailed information of antibodies used in this study is listed in
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