For morphological reconstruction of patch-clamped neurons, 0.2% biocytin (Sigma-Aldrich) was added to the internal solution. Cells were filled for at least 15 min after break-in. Pipettes were then retracted slowly, over 1–2 min, until the recorded membrane fully resealed. Following slice physiology procedures, slices were post-fixed in 4% PFA overnight at 4 °C and then washed with PBS X3 before being incubated with streptavidin-conjugated DyLight 549 or DyLight 405 (Vector Labs) at 1:1,000 dilution for 2 h at room temperature to label cells that were filled with biocytin (2%; Sigma-Aldrich) during patch clamp recordings. Slices were then mounted for microscopy on glass slides using Aquamount (Thermo Scientific) and imaged the next day on Leica TCS SP8 confocal microscope, using a ×40 1.3 NA, oil immersion objective, at ×0.9–1.0 zoom with an xy sampling frequency of approximately 7 pixels per micrometre. Z stacks at 1-μm intervals were serially obtained and z-stack tiling and Leica-based automated stitching were performed to cover the entire dendritic tree of each neuron. The neurons were subsequently semi-manually traced using the neuTube interface40 (link) and SWC files were generated. The files were next loaded into Fiji (ImageJ, version 2.1.0; NIH) plugin SimpleNeuriteTracer41 (link).
Dylight 549
Manufactured by Vector Laboratories
Sourced in United States
DyLight 549 is a fluorescent dye produced by Vector Laboratories. It is designed for use in various biological and research applications that require a fluorescent label. The dye has an excitation wavelength of 542 nm and an emission wavelength of 572 nm.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Aquamount, by Thermo Fisher Scientific (1 mentions)
Biocytin, by Merck Group (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Dylight 594, by Abcam (1 mentions)
Dylight 488, by Abcam (1 mentions)
Fluoview fv10i confocal microscope, by Olympus (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
A1r confocal laser scanning system, by Nikon (1 mentions)
Nis element, by Nikon (1 mentions)
Acti stain 488, by Cytoskeleton (1 mentions)
Prolong glass antifade with nucblue, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Neurobiotin, by Vector Laboratories (1 mentions)
Neurobiotin, by Merck Group (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Dapi containing mounting medium, by Vector Laboratories (1 mentions)
8 well chamber slide, by Thermo Fisher Scientific (1 mentions)
Axioplan 2 microscope, by Zeiss (1 mentions)
7 protocols using dylight 549
1
Morphological Reconstruction of Patch-Clamped Neurons
2
Immunohistochemical Analysis of PI5P4K Isoforms in PVR
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Frozen sections of embedded frozen ERMs from three patients with grade C PVR were prepared as described previously,7 (link) and this research adhered to the Tenets of the Declaration of Helsinki and the protocol approved by the Schepens Eye Research Institute Massachusetts Eye and Ear Institutional Review Board (632172-3). The sections were fixed in 3.7% formaldehyde/PBS for 10 minutes and were then treated for 15 minutes with 0.3% hydrogen peroxide (to remove endogenous peroxidases). Subsequently, the sections were preincubated with 5% normal goat serum in 0.3% Triton X-100/PBS for 20 minutes, then incubated with primary antibodies against PI5P4Kα or PI5P4Kβ (1:100 dilution) for 1 hour or with a normal rabbit IgG. After three washes with PBS, the sections were incubated with fluorescently labeled secondary antibodies, Dylight 549 (Vector Laboratories, Inc., Burlingame, CA, USA) (1:300 dilution in a blocking buffer), for 30 minutes. After three washes with PBS, the slides were mounted with a mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) and photographed under a fluorescence microscope.21 (link),40 (link)
3
Visualization of RNA Synthesis in DAOY Cells
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DAOY cells were seeded in chamber slides (0,3 cm2/well; 5×103 cells/well) and at the indicated time intervals p.i. processed as previously described [47 (link)]. Rabbit anti-POLR1A (Abcam; 1:200) and chicken anti-NS3 (a kind gift from Dr. M. Bloom, NIAID, NIH; 1:5000) antibodies were used. As the secondary antibodies, anti-rabbit DyLight 594 (Abcam; 1:500) and anti-chicken DyLight 488 (Abcam; 1:500), were used. In the case of metabolic labelling of nascent RNA, the Click reaction was performed in situ before the blocking step. 10 μM Picolyl biotin azide was used for the detection of incorporated 5-EU. For subsequent fluorescent labelling, streptavidin conjugated with DyLight 549 was used (VectorLabs; 1:500). Slides were eventually mounted in Vectashield mounting medium (VectorLabs). The Olympus Fluoview FV10i confocal microscope was used for imaging and subsequent export of images was done in FV10-ASW software (v.1.7).
4
Localization of leiomodin-1 in HITC6 cells
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HITC6 cells grown on glass coverslips were fixed with 4% (v/v) paraformaldehyde, permeabilized with 0.5% (v/v) Triton X-100 in phosphate buffered saline (PBS) and blocked with 5% (v/v) normal goat serum. Coverslips were incubated with leiomodin-1 antibody (Proteintech, #15117-1-AP, rabbit polyclonal, 1:3000) overnight at 4 °C followed by incubation with goat anti-rabbit IgG conjugated with DyLight 549 (Vector Laboratories, 1:400). Actin filaments were stained with Alexa Fluor 488-conjugated phalloidin (Acti-stain 488, Cytoskeleton, #PHDG1, 100 nM in PBS). Cells were mounted with ProLong Glass anti-fade with NucBlue (ThermoFisher Scientific). Fluorescent images were acquired with a Nikon A1R confocal laser scanning system using a 60X oil-immersion objective (numerical aperture 1.4) and 405 nm, 488 nm, and 561 nm lasers, generating up to 9 z-slices with a pixel resolution of 100 nm and z-step size of 250 nm using a Galvano scanner. Maximal intensity projection images were rendered with Nikon NIS-Elements.
5
Morphological Examination of L4 Axons
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For morphological examination of L4 axons, 0.5∼1% of neurobiotin (Vector Laboratories) was included in the pipette solution. After whole-cell recordings, the slices containing neurobiotin-loaded cells were fixed by immersion in 4% PFA in 0.2 M phosphate buffer (PB) for 1 h at room temperature, followed by incubation in PB and 5% Triton-X100 (Sigma-Aldrich) for 2 d. Then, slices were incubated with streptavidin-fluorescein (Alexa Fluor 488 conjugate, Thermo Fisher Scientific, or DyLight 549, Vector Laboratories). After three rinses in PBS, the slices were embedded with ProLong Diamond Antifade Mountant (Invitrogen, Thermo Fisher Scientific), coverslipped, and sealed with Paper Bond (Kokuyo). DAPI staining was also performed for identification of the barrel boundary as described above.
6
NF-κB Localization by Immunofluorescence
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For immunofluorescence C3842 and SW1353 cells were seeded into 8 well chamberslides (Thermo) and treated with IL-1β (10 ng/ml) for 120 min, and/or Curcumin (20 µmol/l) as described above. Afterwards, the cells were washed twice with cold PBS and immediately fixed with methanol (−20°C) for 10 min and acetone (−20°) for further 5 min. The slides were stored in PBS at 4°C until further use. For immunostaining the slides were blocked in normal goat serum (10%; Vectorlabs, Burlingame, CA, USA) for 60 min at room temperature. Primary antibody directed against the p65 subunit of NF-κB (1∶100; Cell-Signaling) was performed overnight in a humidified chamber at 4°C. After three washes (5 min) with PBS, a secondary antibody labelled with Dylight 549 (1∶1000; Vectorlabs) was applied. The slides were washed with PBS three times for 5 minutes and mounted in DAPI-containing mounting medium (Vectorlabs). For visualisation and photography a Zeiss Axioplan 2 microscope (Zeiss, Oberkochen, Germany) equipped with an ISIS in-situ imaging system (MetaSystems, Altlussheim, Germany) was used.
7
Immunofluorescent labeling of neural cells
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Following fixation, cell permeabilisation was performed using 0.5% TritonX-100 (Sigma) for 30 min. Following three times of 5-min washes, non-specific binding was blocked with 5% normal goat serum (Dako, Ely, UK) in PBS for 30 min. After another wash step, primary antibodies diluted in PBS (mouse anti-β-tubulin III IgG; 1:400 (Sigma), rabbit anti-glial fibrillary acidic protein (GFAP); 1:300 (Dako)) were applied to gels and incubated overnight at 4°C. Following three times of 10-min washes, secondary antibodies (anti-mouse DyLight 488 and anti-rabbit DyLight 549; 1:300; Vector Laboratories) diluted in PBS were added for 90 min. Hoechst 33258 was also added into the secondary antibody dilutions for cell counting. Omission of primary or secondary antibody was routinely used as a control.
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