Alexa 568 hydrazide was used at—10 mM (Thermo Fisher Scientific), Antisauvagine-30 (aSvg)—150 nM (Tocris), bicuculline—20 μM (Sigma Aldrich), CRF—100 nM (Bachem), NBI 27914 (NBI)—1.2 µM (Tocris), TTX—1 µM (Tocris). Besides Alexa 568, all compounds were dissolved in DMSO prior to dilution into appropriate aqueous buffers/solutions.
Alexa 568 hydrazide
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 568 hydrazide is a fluorescent dye that emits in the orange-red region of the visible spectrum. It is commonly used as a labeling agent for various biomolecules, including proteins, nucleic acids, and polysaccharides, to enable their detection and visualization in biological applications.
Automatically generated - may contain errors
4 protocols using alexa 568 hydrazide
1
Receptor Pharmacology Assay Protocol
2
Visualizing Neuron Morphology Post-Stimulation
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We used microinjection and confocal microscope (Dumitriu et al. 2011) to visualize and quantify the neural morphology at 24 h and 48 h post-stimulation. The sections were selected within the distance of approximately ±0.4 mm anterior-posterior (AP) away from the optic fiber. The injection was done only into the pyramidal neurons from layer 2 -3 of ACC (24a/24b) from both hemispheres. The injection pipettes were pulled from glass capillaries with filament, with a final resistance around 150 MΩ. We filled the pipette with red fluorescent dye solution Alexa 568 hydrazide (#A10441, Thermo Fisher, USA) in filtered 1× PBS (1 : 40). We performed microinjection under the microscope of a patchclamp set-up. During injection, we penetrated the pipette tip into the soma and switched on the current to -20 pA to drive the dye diffusion for 20 min. Later we switched off the current but left the pipette tip inside the soma for another 5 min to fill the dendrite and spines. All the sections were retrieved and covered with Vectashild H-1000 (Vector Laboratories, Germany) for confocal microscope imaging. We checked all the injected neurons for YFP signal; only neurons with YFP signal were identified as pyramidal neurons and selected for further analysis.
3
Visualizing Neuronal Morphology in ACC
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We used microinjection and confocal microscope (Dumitriu et al. 2011) to visualize and quantify the neural morphology at 24 h and 48 h post-stimulation. The sections were selected within the distance of approximately ±0.4 mm anterior-posterior (AP) away from the optic fiber. The injection was done only into the pyramidal neurons from layer 2 -3 of ACC (24a/24b) from both hemispheres. The injection pipettes were pulled from glass capillaries with filament, with a final resistance around 150 MΩ. We filled the pipette with red fluorescent dye solution Alexa 568 hydrazide (#A10441, Thermo Fisher, USA) in filtered 1× PBS (1 : 40). We performed microinjection under the microscope of a patch-clamp set-up. During injection, we penetrated the pipette tip into the soma and switched on the current to -20 pA to drive the dye diffusion for 20 min. Later we switched off the current but left the pipette tip inside the soma for another 5 min to fill the dendrite and spines. All the sections were retrieved and covered with Vectashild H-1000 (Vector Laboratories, Germany) for confocal microscope imaging. We checked all the injected neurons for YFP signal; only neurons with YFP signal were identified as pyramidal neurons and selected for further analysis.
4
Neurochemical Modulation Assay
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The used dilutions: Alexa 568 hydrazide -10 mM (Thermo Fisher Scientific), Antisauvagine-30 (aSvg) -150 nM (Tocris), bicuculline -20 μM (Sigma Aldrich), CRF -100 nM (Bachem), NBI 27914 (NBI) -1.2 µM (Tocris), TTX -1 µM (Tocris). Besides Alexa568, all drugs were dissolved in DMSO prior added into used solutions.
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