AlexaFluor-594 hydrazide, AlexaFluor-594 dextran (10,000 MW), Fluo-4 and Fluo-4FF pentapotassium salts were purchased from Invitrogen. CsCl and NaHCO3 were obtained from ACP Chemicals, tetrodotoxin (TTX) from Alomone Labs, N-ethyllidocaine bromide (QX-314, Br-) and bicuculline from Tocris Bioscience, SP20 antibody from Santa Cruz Biotechnology (Cat# sc-65512, Lot# K1407, RRID:AB_1129364), paraformaldehyde from Electron Microscopy Sciences. We ordered NaCl, KCl, glucose, NaH2PO4, Na-pyruvate, myo-inositol, l -ascorbic acid, MgCl2, CaCl2, K-gluconate, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), HEPES, phosphocreatine, Na-ATP, Na-GTP, phosphocreatine di(tris) salt, tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP) and Strychnine from Sigma-Aldrich.
Alexa fluor 594 hydrazide
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor® 594 Hydrazide is a fluorescent dye used in various applications in life science research. It is a hydrazide derivative of the Alexa Fluor® 594 fluorophore, which has an excitation maximum of 590 nm and an emission maximum of 617 nm. The hydrazide group allows for covalent labeling of biomolecules, such as proteins and carbohydrates, through reaction with aldehyde or ketone groups.
Automatically generated - may contain errors
Lab products found in correlation
Phosphocreatine, by Merck Group (1 mentions)
Strychnine, by Merck Group (1 mentions)
Na pyruvate, by Merck Group (1 mentions)
K gluconate, by Merck Group (1 mentions)
Myo inositol, by Merck Group (1 mentions)
Na atp, by Merck Group (1 mentions)
Fluo 4, by Thermo Fisher Scientific (1 mentions)
Ethylene glycol bis, by Merck Group (1 mentions)
Alexafluor 594 dextran, by Thermo Fisher Scientific (1 mentions)
Na gtp, by Merck Group (1 mentions)
Mgcl2, by Merck Group (1 mentions)
4 aminopyridine 4 ap, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Glucose, by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Cacl2, by Merck Group (1 mentions)
Nah2po4, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
10 protocols using alexa fluor 594 hydrazide
1
Reagents for Neurophysiological Experiments
2
Quantifying Phagosome-Lysosome Fusion by FRET
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cDC1 sorted from Flt3L-cultured BM cells were incubated with 100 μg/ml Alexa Fluor 594-Hydrazide (Cat#A10438; Invitrogen) for 4 h. Then cells were washed with PBS and were incubated without tracer for a further 12 h to allow accumulation of the dye in lysosomes. Cells were resuspended in new DC medium supplemented with Alexa Fluor 488-labeled HKLM-OVA. Then cells were rinsed and resuspended in medium at 37 °C for analysis by spectrofluorometer. As lysosomes fuse with phagosomes, the donor Alexa Fluor 488 comes into close proximity with acceptor Alexa Fluor 594, causing FRET. The degree of lysosomes fused with phagosomes could be calculated from the ratio between the FRET-generated emission at 620 nm and the donor emission at 510 nm using the equation RFU = FRT/DRT-FBO/DBO, where RFU represented relative FRET units, FRT represented FRET-generated fluorescent emission, DRT represented Donor emission, FBO represented FRET signal contribution of HKLM-OVA alone and DBO represented Donor emission of HKLM-OVA alone. Fluorescent measurements were taken every 1 min for 3 h.
3
Single-neuron Labeling in Xenopus Olfactory Bulb
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The granule cell in the Xenopus olfactory bulb was labeled with plasmid DNA by single-cell electroporation in vivo20 (link)37 (link). 0.5–0.8 μg/μl GFP plasmid mixed with 0.05 mM Alexa Fluor® 594 Hydrazide, a red fluorescent dye (A10438, Invitrogen, USA), were co-electroporated into the olfactory granule cell layer. After 2 days, tadpoles with a single spiny neuron expressing GFP throughout their cell bodies, dendrites, and spines were selected for in vivo imaging.
4
Patch-Clamp Recording of Fluorescent Neurons
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The extracellular perfusing solution was continuously bubbled with 95% O 2 -5% CO 2 and had the following composition (in mM): 125 NaCl, 25 NaHCO 3 , 2.5 KCl, 2.5 CaCl 2 , 1 MgCl 2 , and 10 glucose, pH 7.4. The recording pipettes were filled with an intracellular solution of the following composition (in mM): 125 K-gluconate, 5 KCl, 8 NaCl, 0.2 EGTA, 10 Hepes, 4 MgATP, and 0.4 Na 3 GTP (pH was adjusted to 7.3 with KOH). The pipette solution also contained Alexa Fluor 594 hydrazide (40 -60 µM), as a sodium salt (Invitrogen / Molecular Probes).
Patch pipettes were pulled from thick-walled borosilicate glass (outer diameter, 1.5 mm; inner diameter, 0.86 mm; BF150-86-10; Sutter Instrument) and the open tip resistance was 7 -12 M" when filled with the intracellular solution. Whole cell voltage clamp recordings were performed with an EPC10-triple amplifier controlled by PatchMaster software (HEKA Elektronik, Lambrecht/Pfalz, Germany). During image acquisition, cells were voltage clamped at a holding potential of -60 mV (corrected for the liquid junction potential).
Patch pipettes were pulled from thick-walled borosilicate glass (outer diameter, 1.5 mm; inner diameter, 0.86 mm; BF150-86-10; Sutter Instrument) and the open tip resistance was 7 -12 M" when filled with the intracellular solution. Whole cell voltage clamp recordings were performed with an EPC10-triple amplifier controlled by PatchMaster software (HEKA Elektronik, Lambrecht/Pfalz, Germany). During image acquisition, cells were voltage clamped at a holding potential of -60 mV (corrected for the liquid junction potential).
5
Whole-Cell Patch-Clamp of Layer 3 Neurons
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Layer 3 neurons from acute cortical slices (P24 rat) were whole-cell patch-clamped with borosilicate glass electrodes (3–4 MΩ). The intracellular recording solution consisted of the following (in mm ): 113 K-gluconate, 2 MgCl2, 8 Na-gluconate, 3 KCl, 1 K2-EGTA, 4 K2-ATP, and 0.3 Na3-GTP at pH 7.25 with 10 HEPES. The solution also contained 50 µm AlexaFluor 594 hydrazide (ThermoFisher) to visualize the morphology of the dendritic arbor. The example cell was dialyzed with dye for 30 min before the patch electrode was slowly withdrawn before imaging. Images were post-processed in Fiji to subtract background using rolling ball radius, and enhance connectivity while removing speckles using the Gaussian Blur 3D filter and smooth functions.
6
Fluorescent Labeling of CA1 Neurons
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Mice were deeply anesthetized with ketamine, and perfused with 0.9% w/v Sodium Chloride, then perfused with 4% paraformaldehyde (PFA) in phosphate buffer (PB). The brain tissues were removed and post-fixed in 4% PFA in PB for 30 min. Using a vibratome, 100 µm coronal sections were sliced and stored in 1× dPBS. Alexa Fluor® 594 Hydrazide (Thermo Fisher Scientific A10438) was injected into CA1 pyramidal neurons to follow neuronal projections. Injected slices were post-fixed for 15 min on 4% PFA in PB prior to mounting with Aqua-Poly/Mount (Polysciences Inc. 18606-20).
7
Pharmacological Agents Sourcing Protocol
8
PEG Hydrazide Fluorescent Labeling
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Alcohol-terminated 8-arm PEG (molecular weight (MW 10 kDA) was purchased from Creative PEGWorks (North Carolina, USA). Alexa Fluor® 594 NHS Ester (Succinimidyl Ester) and Alexa Fluor® 594 Hydrazide were purchased from Thermo Fisher Scientific. All other chemicals were purchased from Sigma-Aldrich and were used as received without further purification.
9
Pharmacological Modulation of Neural Signaling
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The following concentrations of each pharmacological reagent were used for experiments as indicated in the text: tetrodotoxin citrate (TTX, 1 µM, Hello Bio); carbenoxolone disodium (CBX, 50 µM, Tocris Bioscience); R(+)-baclofen hydrochloride (5–100 µM, Sigma-Aldrich); (1S,3R)-ACPD (t-ACPD, 5–100 µM, Tocris); LY379268 disodium salt (100 µM, Tocris); Alexa Fluor 594 hydrazide (0.1–2 µM, Thermo Fisher Scientific); RuBi GABA trimethylphosphine (RuBi-GABA-Pme3, 300 µM, Tocris); RuBi–Glutamate (300 µM, Tocris); CGP 55845 hydrocholoride (10 µM, Tocris); and LY341495 (10 µM, Tocris).
10
Patch-Clamp Electrophysiology in Brain Slices
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For the recordings, we placed the slices in constantly renewed CO2 sparged extracellular solution (in mm as follows: 125 NaCl, 25 NaHCO3, 25 glucose, 2.5 KCl, 2 CaCl2, 1.25 NaH2PO4, 1 MgCl2, sparged with 95% O2/5% CO2). Pipettes with 1–2 μm opening diameter and 4–10 m Ω resistance depending on cell size were pulled from 2.0-mm-diameter borosilicate glass capillaries (Hilgenberg) using a DMZ universal puller (Zeitz Instruments). We filled pipettes with an intracellular solution containing the following (in mm : 130 KMeSO4, 10 HEPES, 7 KCl, 2 ATP-Na, 2 ATP-Mg, 0.5 GTP, 0.05 EGTA, adjusted to pH 7.4 with KOH, osmolarity 293 mOsm). For the visualization of cells during recording, we added 0.8 mm of fluorescent dye Alexa-488 or AlexaFluor-594 hydrazide (Thermo Fisher Scientific) to the intracellular solution, whereas for post hoc neuron reconstruction, we added biocytin (Sigma-Aldrich) at a concentration of 2 mg/ml as histological stain, filling the cells during 2–25 min of recording.
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