Following recovery, cells were extensively washed with PBS and permeabilized with PBS containing 0.1% Triton X-100. Subsequently, samples were incubated overnight at 4°C with anti-phospho-p38 MAPK (pThr180/pTyr182) monoclonal rabbit antibody, anti-phospho-Jun-N-terminal kinase (pThr183/pTyr185) monoclonal rabbit antibody or anti-phospho-Erk1/2 (pThr202/pTyr204) monoclonal rabbit antibody, all obtained from Cell signaling Technology (http://www.cellsignal.com/ ) followed by incubation with the secondary antibody anti-rabbit AlexaFluor488 (Invitrogen, Carlsbad, CA) for 1 h at room temperature and counterstained with DAPI. Confocal images were obtained on a Leica TCS SP2 system equipped with 405-nm UV, 488-nm argon, and 543-HeNe lasers (Leica, Mannheim, Germany) and processed using ImageJ (National Institutes of Health, Bethesda, MD) software for constructing total fluorescence signal and automated analysis of nuclear versus cytoplasmic signal.
Tcs sp2 system
Manufactured by Leica
Sourced in Germany
The TCS SP2 system is a confocal laser scanning microscope developed by Leica. It is designed to provide high-resolution imaging of samples by using a focused laser beam to scan the specimen and collect the emitted fluorescence or reflected light. The core function of the TCS SP2 system is to enable detailed, non-invasive imaging of various biological and material samples.
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Lab products found in correlation
Dmire2 inverted microscope, by Leica (4 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Lsm software, by Zeiss (1 mentions)
Attofluor holder, by Thermo Fisher Scientific (1 mentions)
Hcx pl apo cs 63, by Leica (1 mentions)
Metamorph software, by Universal Imaging (1 mentions)
Pg 21, by Merck Group (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Aristoplan, by Leica (1 mentions)
Tsc sp5 system, by Leica (1 mentions)
Draq5, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Normal goat serum (ngs), by MP Biomedicals (1 mentions)
Donkey anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 system, by Leica (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
17 protocols using tcs sp2 system
1
Phospho-specific Antibody Imaging Assay
2
Calu-3 Cell Actin Cytoskeleton Imaging
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Calu-3 cells grown to confluence on Transwell® inserts as described above, were incubated in the presence and absence of Solutol® HS15 (5.2 mM and 0.1 mM) for 3 h at 37°C. Following the incubation cells were fixed with paraformaldehyde at room temperature for approximately 10 min, washed with PBS and permeabilised using Triton X-100 (3.1 mM in PBS) for approximately 10 min. After a series of washing stages using PBS, 1% w/v bovine serum albumin (BSA, in PBS) was applied for approximately 1 h. Thereafter, a 0.17 μM Alexa Fluor-546 Phalloidin in 1% w/v BSA solution was applied to each well and incubated for 20 mins, following the manufacturer’s instructions. Following incubation, each insert was extensively washed with PBS and filter membrane excised and mounted (using DABCO mounting medium) on glass slides for confocal imaging. Cells were imaged using a Leica TCS SP2 system mounted on a Leica DMIRE2 inverted microscope.
3
Confocal Imaging of Immunofluorescence Samples
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Immunofluorescence was performed as described previously using FITC‐, or Alex 488‐conjugated secondary antibodies (Abcam).22 Confocal microscopy was performed at room temperature (RT) on a microscope (model LSM 510; Carl Zeiss MicroImaging, Inc) and a TCS SP2 system (Leica; 63 oil immersion objectives, NA 1.4). Immunofluorescence images were taken sequentially, and parameters were adjusted so that all light intensities were in the recording range. Confocal stacks were acquired with a slice thickness of 300 nm. Micrographs were derived as indicated in the figure legends from single confocal planes, several merged confocal planes, vertical sections generated from confocal stacks with the LSM software (Carl Zeiss MicroImaging, Inc), or as a merged image of several vertical sections generated by reslicing of a confocal stack with Image J software (W. Rasband, National Institutes of Health, Bethesda, MD). Pictures were arranged with Adobe Photoshop and Adobe Illustrator. Experiments were performed at least in triplicate, and representative images of highly expressing cells are shown. Similar experiments were performed using PMVECs transfected with siRNAs or treated with inhibitor.
4
Confocal Microscopy Imaging of Fluorescent Proteins
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Confocal microscopy experiments were performed on a Leica TCS SP2 system, equipped with a HCX PL APO CS 63.0 × 1.32 oil objective. Coverslips with cells expressing the desired constructs were mounted using an Attofluor holder (Molecular Probes) and cells were maintained in the imaging buffer. TFP was excited at 458 nm and fluorescence intensities were recorded from 465–550 nm. Images were taken with 512 × 512 pixel format, 400 Hz, line average 4, frame average 3.
5
Sperm Nuclear Migration and Mitochondria Dynamics
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To visualize the process of sperm nuclear migration and fluorescence of DAPI-stained nuclei, cells were examined using a Leica epifluorescence inverted microscope with a ×63 objective, equipped with a cooled CCD (RTE/ CCD-1300-Y/HS, Roper Scientific Inc.). Digital images from CCD were captured on a computer equipped with the MetaMorph software (Universal Imaging Corporation Inc.).
To visualize the movement of mitochondria in tobacco, the tobacco plants expressing a mitochondrial marker, p35S::coxlV-GFP, were used. Tobacco leaves were observed by confocal laser scanning microscopy on a TCS SP2 system (Leica). The 488 nm line of an argon/krypton laser was used for excitation. The fluorescence light longpass was filtered from 515 to 550 nm before being detected by a photomultiplier.
To visualize the movement of mitochondria in tobacco, the tobacco plants expressing a mitochondrial marker, p35S::coxlV-GFP, were used. Tobacco leaves were observed by confocal laser scanning microscopy on a TCS SP2 system (Leica). The 488 nm line of an argon/krypton laser was used for excitation. The fluorescence light longpass was filtered from 515 to 550 nm before being detected by a photomultiplier.
6
Androgen Receptor Localization in Cells
7
Microscopic Imaging of Stained Tissue
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A Leica bright-field microscope (Leica Aristoplan, with digital camera using Leica 1000 software) was used to assess and acquire images from H&E-stained and peroxidase-stained sections. Immunofluorescent stains were analyzed and images acquired and processed using a Leica TCS SP2 system, a Leica TSC SP5 system, and UV CLSM acquisition software and a Leica DM600B UV microscope with FW4000 acquisition software. In the CLSM experiments, the red and green channels were imaged sequentially.
8
Subcellular Localization of AR Variants
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Subcellular localization of AR proteins was analyzed by confocal fluorescence microscopy. The pTurboFP635-AR-V7 and pTurboFP635-ARv567es plasmids were generated by cloning the cDNA fragments for AR-V7 and ARv567es, respectively, into the pCMV-TurboFP635 vector. COS-7 cells were transfected with indicated plasmids and cultured in phenol red-free RPMI-1640 supplemented with 10% cs-FBS. At 40 hr after transfection, cells were pre-treated with or without 10 nM docetaxel for 6 hr, followed by treatment with or without 1 nM R1881 for 4 hr. COS-7 cells were subsequently fixed with 2% paraformaldehyde, and the nuclei were stained with 2.5 μM DRAQ5 (Cell Signaling). Confocal images were obtained by using a Leica TCS SP2 system with a 63X oil-immersion objective on a Z-stage, and an average of 6 fields with ∼10 cells per field were captured for each group. Data quantitation was performed as described [44 (link)].
9
Evaluating Solutol HS15 Effects on Calu-3 Tight Junctions
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Confluent Calu-3 cell layers grown on Transwell® inserts were incubated with 0.1 and 5.2 mM Solutol® HS15 in HBSS:HEPES for 3 h. HBSS:HEPES (25 mM, pH 7.4) alone was used as the negative control. Following the incubation, cells were fixed with 4% w/v paraformaldehyde at room temperature for approximately 10 min, washed with PBS and permeabilised using Triton X-100 (3.1 mM in PBS) for approximately 10 min. After a series of washing stages using PBS, 1% w/v bovine serum albumin (BSA) in PBS solution was applied for approximately 1 h and then removed. Thereafter, BSA/PBS solution was replaced with mouse, anti-human ZO-1 (primary) antibody, in 1% w/v BSA/PBS, at 10 μg/ml and incubated for 1 h. Cells were washed and FITC-labelled goat, anti-mouse (secondary) antibody, diluted according to manufacturer’s instructions in 1% w/v BSA/PBS, was applied for 1 h. Each insert was then washed with PBS extensively and the filter membrane excised and mounted (using DABCO mounting medium) on glass slides for confocal imaging. Cells were imaged using a Leica TCS SP2 system mounted on a Leica DMIRE2 inverted microscope.
10
Immunolocalization of FcRn in Caco-2 Monolayers
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Polarised Caco-2 monolayers were fixed in paraformaldehyde, washed with PBS and permeabilised by incubating with Triton X-100 (1% v/v in PBS) for approximately 10 min.
Cells were washed again and incubated for 1 hour with BSA in PBS (1% w/v). Goat, antihuman FcRn primary antibody was diluted to 10 µg/ml in 1% w/v BSA/PBS and incubated with the cells for 30-60 min at room temperature. Cells were then washed extensively and treated with donkey, anti-goat TRITC-IgG (secondary antibody, diluted according to supplier's instructions in 1% BSA/PBS) for 30-60 min. Thereafter, cells were washed extensively and filters excised and mounted on a glass slide (using a DAPI-containing ProLong ® Gold antifade mounting medium) for confocal imaging. Control experiments were conducted where the cells were treated with the secondary antibody only. Confocal imaging was conducted using a Leica TCS SP2 system mounted on a Leica DMIRE2 inverted microscope.
Cells were washed again and incubated for 1 hour with BSA in PBS (1% w/v). Goat, antihuman FcRn primary antibody was diluted to 10 µg/ml in 1% w/v BSA/PBS and incubated with the cells for 30-60 min at room temperature. Cells were then washed extensively and treated with donkey, anti-goat TRITC-IgG (secondary antibody, diluted according to supplier's instructions in 1% BSA/PBS) for 30-60 min. Thereafter, cells were washed extensively and filters excised and mounted on a glass slide (using a DAPI-containing ProLong ® Gold antifade mounting medium) for confocal imaging. Control experiments were conducted where the cells were treated with the secondary antibody only. Confocal imaging was conducted using a Leica TCS SP2 system mounted on a Leica DMIRE2 inverted microscope.
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