Coverslips containing neuronal cultures were transferred to a recording chamber for live imaging of the TEpacVV biosensor (Klarenbeek et al., 2011 (link)) using a Leica TCS SP8 MP confocal microscope. Excitation of the mTurquoise FRET donor was achieved with a 442 nm diode laser which was paired with collection of bandpass emission filtration at 3.5 Hz simultaneously from 465–505 nm (mTurquoise FRET donor) and 525–600 nm (Venus FRET acceptor). Image stacks containing XYZ planes were acquired at 10 s intervals through a 25x objective lens. Quantification of fluorescence intensity was performed on neuronal cell bodies using ImageJ to calculate FRET from the inverse ratio of donor:acceptor. Absolute cAMP values were determined from interpolation of a cAMP standard curve in permeabilized CAMPER neurons (Muntean et al., 2018 (link)). Dopamine and adenosine were added in phasic puffs in the pH 7.2 recording buffer which consisted of (in mM): CaCl2 (1.3), MgCl2 (0.5), MgSO4 (0.4), KH2PO4 (0.4), NaHCO3 (4.2), NaCl (138), Na2HPO4 (0.3), D-Glucose (5.6), and HEPES (20). In dMSNs D1R stimulates and A1R inhibits cAMP whereas in iMSNs A2AR stimulates and D2R inhibits cAMP. Therefore, sign of cAMP response classified neurons as dMSN or iMSN without the need for additional reagents to isolate the cAMP signaling pathway.
Tcs sp8 mp confocal microscope
Manufactured by Leica
Sourced in Germany
The Leica TCS SP8 MP confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a multipoint scanning technology that enables fast and efficient data acquisition. The microscope is equipped with a range of laser excitation sources and specialized detectors, providing users with a versatile platform for various research and analysis needs.
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Lab products found in correlation
Imagequant, by GE Healthcare (3 mentions)
Volocity, by PerkinElmer (3 mentions)
Las af lite software, by Leica (1 mentions)
Click it edu alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Rm2245 microtome, by Leica (1 mentions)
Axio imager m1, by Zeiss (1 mentions)
Technovit 7100 resin, by Kulzer (1 mentions)
Application suite x software, by Leica (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Alexa 568 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ab3623, by Merck Group (1 mentions)
Prolong antifade, by Thermo Fisher Scientific (1 mentions)
Celltracker deep red, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Click it tunel alexa fluor 488 imaging assay, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Lsm 700, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Dako fluorescent mounting medium, by Agilent Technologies (1 mentions)
20 protocols using tcs sp8 mp confocal microscope
1
FRET-based cAMP Signaling in MSNs
2
Confocal Microscopy of Fluorescent Cells
3
FRET-based cAMP Signaling in MSNs
4
Quantification of Vessel-Associated GABAergic Interneurons
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Quantification of vessel-associated GABAergic interneurons was performed after double immunostaining of P2 cortical slices with GABA and CD31 antibodies (Supplementary Table 1). Images were acquired and saved in TIFF format using a Leica TCS SP8 MP confocal microscope. Image resolution was 1024 × 1024 pixels and z stacks were done with a step ranging from 0.25 to 0.5 µm (Supplementary Fig. 2d). X/Z as well as Y/Z sections of the acquired stacks were done using the IMARIS imaging software (Bitplane, Zurich, Switzerland) to validate the vessel–neuron interactions (Supplementary Fig. 2a–c). Images were subsequently opened in Leica LAS AF Lite software to quantify the distance between the outer part of the neuron and the outer part of the vessel (Supplementary Fig. 2e). Distances below 15 µm were considered as representative of a vessel association (Supplementary Fig. 2e). Finally, the vessel density was quantified in the superficial and deep cortical layers, and this parameter was used to balance the vessel-associated vs not-associated proportion of GABA interneurons. The quantification of the PMR and cortical layer thicknesses in t-PA null mice and in Grin1lox/lox/VeCadCre mice was also performed using the Leica LAS AF Lite software.
5
Cell Proliferation Quantification by Click-iT EdU Assay
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Cell proliferation was measured using Click-iT® EdU Alexa Fluor 647 according to the manufacturer’s instructions (C10340, Thermo Fisher Scientific, Waltham, MA). Briefly, Click-iT® EdU Alexa Fluor 647 is a modified thymidine analogue EdU (5-ethynyl-2′-deoxyuridine, a nucleoside analog of thymidine) that is incorporated into newly synthesized DNA. The EdU is fluorescently labeled with a photostable Alexa Fluor® dye during the click reaction. Uninjured and injured cortical cells co-cultured with and without microglia for 2 DIV (days in vitro) were fixed in 3.7% formaldehyde in PBS for 15 min at RT. Fixed cells were washed twice with 1 ml of 3% BSA in PBS. Cells were permeabilized in 0.5% Triton®x-100 for 20 min at RT, washed and 1X Click-iT® EdU reaction cocktail was added for 30 min at RT. The reaction cocktail was removed, cells were washed in 3% BSA and PBS, counterstained with DAPI, mounted and imaged for analysis. Imaging was performed using IBIF Leica TCS SP8 MP Confocal Microscope at 20 × magnification. Experiments were performed in triplicate with at least 300 cells counted manually per experiment for each condition. Volocity (PerkinElmer, USA) and ImageQuant (GE Healthcare, USA) software were used for image analysis and presentation.
6
mRNA Expression Profiling in Striatal Neurons
7
Labeling Mitotically Active Cells in Cochlea
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To label mitotically active cells, a single, subcutaneous injection of the thymidine analog 5-ethynyl-2′-deoxyuridine (EdU; 50 mg/kg) in DMSO was administered to DN-CBRb+/ROSA-CAG-rtTA+ and DN-CBRb+/ROSA-CAG-rtTA- mice 4 h before tissue harvesting. EdU incorporation into DNA of whole-mount cochlea was detected using the Click-iT EdU Alexa 488 Fluor Imaging kit (Invitrogen) and double stained with DAPI following the manufacturer’s instructions and experimental procedures previously described (Kaiser et al., 2009 (link); Rocha-Sanchez et al., 2011 (link)). Samples were imaged using a Leica TCS SP8 MP confocal microscope.
8
Visualizing Plant Cell Structures
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Ovaries and seeds were fixed under a vacuum in FAA solution (5% v/v formaldehyde, 5% v/v acetic acid, 63% v/v ethanol). The samples were then dehydrated through a graded ethanol series and embedded in Technovit 7100 resin (Kulzer, https://www.kulzer-technik.de ), according to the manufacturer’s instructions. Sections (2-µm thick) were cut using an RM2245 microtome (Leica, http://www.leica-microsystems.com ) and stained with 0.02% toluidine blue. To visualize cell walls and starch granules, the sections were stained with 1% w/v safranin in 50% ethanol and counterstained with 1% w/v iodine and 1% w/v potassium iodide. Protein bodies were identified by staining with 0.01% w/v Coomassie Brilliant Blue R-250. Images of stained sections were captured using an Axioimager M1 (Zeiss, http://www.zeiss.com ). Propidium iodide staining was performed as previously described (Sekine et al., 2013 (link)). A series of optical images was obtained under a TCS-SP8 MP confocal microscope (Leica).
9
Fluorescence Recovery After Photobleaching
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Specimens were mounted on 10% agarose pads containing 20mM sodium azide and 10mM levamisole in M9. FRAP was performed using Leica Application Suite X software FRAP module on a Leica TCS SP8 MP confocal microscope. For 1.5-fold embryos and L1 larvae, a 1 μm X 1 μm bleach ROI was defined within the wizard, and mean fluorescence intensity measurements within the ROI were taken at specified intervals. The following experimental time-course was used: 20 pre-bleach frames every 0.4 seconds, 5 bleach frames every 0.4 seconds, and 90 post-bleach frames every 2.0 seconds. Laser intensity during bleach was set to 70.0%, while pre- and post-bleach laser intensity varied from 0.25% to 1.50% on a specimen-by-specimen basis. A pinhole size of 3.0 (units) was used for all FRAP experiments. For L4 larvae, bleach laser intensity was increased to 100%, the number of bleach frames was increased to 10, and the ROI size was increased to 3 μm X 3 μm.
FRAP plots were created and analyzed in Prism, where one-phase association curves derived from the model Y = Y0 + (Plateau—Y0)*(1 –e^(-Kx)) were fitted to the data. For statistical tests, mobile fractions and recovery half-times were derived from one-phase association curves fitted to individual experiments. Mobile fraction = Plateau-Y0; t1/2 = ln(2)/K, where K is the recovery rate constant.
FRAP plots were created and analyzed in Prism, where one-phase association curves derived from the model Y = Y0 + (Plateau—Y0)*(1 –e^(-Kx)) were fitted to the data. For statistical tests, mobile fractions and recovery half-times were derived from one-phase association curves fitted to individual experiments. Mobile fraction = Plateau-Y0; t1/2 = ln(2)/K, where K is the recovery rate constant.
10
In Situ Confocal Imaging of Cancer Cells
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The clusters formed in the device were directly placed on the glass slide and imaged in situ via Leica TCS SP8 MP confocal microscope (Germany) using the 63x objective [34] . Images were captured at each channel's proximal, medial, and distal parts to ensure uniform outcomes. The image processing, cluster size analysis, and the density of fluorescence signals were carried out using the ImageJ software. Using ImageJ for analysis, we enumerated only cancer cells within the area range of 50 – 300 μm2 and circularity 0.3 – 1.0 to ensure that smaller bacteria were excluded from the count.
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