Drugs were freshly prepared from stock solutions and diluted in aCSF. Brain slices were then suprafused with aCSF. 2-amino-5-phosphopentanoate (DL-AP5) and 3,5-dihydroxyphenylglycine (DHPG) were both purchased from Abcam (UK) and prepared in distilled water. Here, 30 µM DL-AP5 was freshly prepared from 1 mM stock solution, and 40 µM DHPG and 80 µM LY367385 were also prepared from 1 mM stock solution.
Dl ap5
Manufactured by Abcam
Sourced in United Kingdom
DL-AP5 is a potent, selective and competitive NMDAR antagonist. It acts by blocking the binding of glutamate and glycine to the NMDAR complex.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 555 maleimide, by Thermo Fisher Scientific (4 mentions)
Alexa 647 maleimide, by Thermo Fisher Scientific (4 mentions)
Lauryl maltose neopentyl glycol, by Anatrace (2 mentions)
Cholesteryl hydrogen succinate, by MP Biomedicals (2 mentions)
Neurobiotin, by Vector Laboratories (2 mentions)
Cgp55845, by Abcam (2 mentions)
Tetrodotoxin, by Abcam (2 mentions)
Picrotoxin, by Bio-Techne (1 mentions)
Optoled light source, by Cairn Research (1 mentions)
Prime95b 22mm, by Teledyne (1 mentions)
Protease inhibitor mixture cocktail, by Roche (1 mentions)
Streptavidin alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Anti h3k4me1, by Abcam (1 mentions)
Protein a g beads, by GE Healthcare (1 mentions)
Anti sirt1, by ABclonal (1 mentions)
Anti hdac1, by ABclonal (1 mentions)
Anti h3k9me2, by Abcam (1 mentions)
Anti rest, by Santa Cruz Biotechnology (1 mentions)
Anti ezh2, by Proteintech (1 mentions)
Anti cdyl, by Merck Group (1 mentions)
8 protocols using dl ap5
1
Pharmacological Modulation of Synaptic Responses
2
Transient Transfection and Labeling of HEK293T Cells for smFRET
3
Transient Transfection and Labeling of HEK293T Cells for smFRET
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HEK293T cells were transiently transfected with GluN1*F554C and GluN2A* DNA at a mass ratio of 2.5:7.5 μg per 10 cm dish. An equivalent mass of GluN1*F554C-TS was used in place of GluN1*F554C for experiments involving it. 300 μM DL-AP5 (abcam) and 30 μM DCKA (abcam) were present during transfection to limit excitotoxicity. One day post-transfection, cells from two 10-cm dishes were harvested and labeled for 1 hour at room temperature with 150 nM of donor fluorophore Alexa 555 maleimide (ThermoFisher) and 600 nM of acceptor fluorophore Alexa 647 maleimide (ThermoFisher) in 3 mL extracellular buffer. After washing, labeled cells were then solubilized for 1 hour at 4°C in buffer containing phosphate-buffered saline, 1% lauryl maltose neopentyl glycol (Anatrace), 2 mM cholesteryl hydrogen succinate (MP Biomedicals), and protease inhibitor (Pierce). Unsolubilized debris were then spun down for 1 hour at 100,000 × g at 4°C, and the supernatant used as the smFRET sample.
4
Nmdar Labeling and Functional Characterization
5
Glutamate Imaging in Neuronal Networks
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SF-iGluSnFR fluorescence imaging experiments were performed on an inverted Olympus IX71 microscope equipped with a Prime95B 22MM back-illuminated CMOS camera (Teledyne Photometrics) using a 60x oil-immersion objective (1.35 NA) with resulting image pixel size 183.3 nm. SF-iGluSnFR fluorescence was recorded using a 470-nm excitation light-emitting diode (OptoLED Light Source, Cairn Research) and a 500–550 band-pass emission filter. Image acquisition was performed using μManager software51 (link). Experiments were conducted in a custom-made open laminar flow perfusion chamber (volume 0.35 ml, perfusion rate ~1 ml/min) at 23–25 °C.
The imaging extracellular solution contained (in mM): 125 NaCl, 26 NHCO3, 12 Glucose, 1.25 NaH2PO4, 2.5 KCl, 2 CaCl2, 1.3 MgCl2 (bubbled with 95% O2 and 5% CO2, pH 7.4). To ensure that recorded SF-iGluSnFR responses originate only from the stimulated axon, we suppressed recurrent activity in the neuronal network by blocking postsynaptic ionotropic glutamate and GABA receptors with (in µM) 50 DL-AP5 (Abcam), 10 NBQX (Abcam), and 50 Picrotoxin (Tocris Bioscience).
The imaging extracellular solution contained (in mM): 125 NaCl, 26 NHCO3, 12 Glucose, 1.25 NaH2PO4, 2.5 KCl, 2 CaCl2, 1.3 MgCl2 (bubbled with 95% O2 and 5% CO2, pH 7.4). To ensure that recorded SF-iGluSnFR responses originate only from the stimulated axon, we suppressed recurrent activity in the neuronal network by blocking postsynaptic ionotropic glutamate and GABA receptors with (in µM) 50 DL-AP5 (Abcam), 10 NBQX (Abcam), and 50 Picrotoxin (Tocris Bioscience).
6
Nmdar Labeling and Functional Characterization
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HEK293T cells were plated into 30 mm dishes and transfected (jetPrime, PolyPlus) with GluN1*F554C and GluN2A* constructs at a mass ratio of 1.5:4.5 μg per 20 ml of media. 300 μM DL-AP5 (abcam) and 30 μM DCKA (abcam) were present in the media and recordings were performed 24 to 48 hours post-transfection. Prior to recording, cells were incubated in 150 nM Alexa 555 maleimide (ThermoFisher) and 600 nM Alexa 647 maleimide (ThermoFisher) for at least 1 hour to mimic smFRET labelling conditions. Outside-out patches were excised and piezo-driven solution exchange was performed as outlined elsewhere51 (link). The external solution was (in mM) 150 NaCl, 20 HEPES, 10 Tricine, 1 CaCl2, and 0.1 glycine, pH 7.4 (NaOH). The pipette solution was 135 CsF, 33 CsOH, 11 EGTA, 10 HEPES, 2 MgCl2 and 1 CaCl2, pH 7.4. Lifted whole cell recordings were performed as described elsewhere52 (link) using the same solutions as above with the addition of 2.5 mM KCl to the external solution.
7
Comprehensive Antibody Panel for Epigenetic Regulation
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Commercial antibodies used were: anti-FLAG (Sigma), anti-CDYL (Sigma, HPA035578), anti-REST (Santa Cruz, sc-25398), anti-EZH2 (Proteintech, 21800-1-AP), anti-HDAC1 (sc-7872), anti-HDAC2 (sc-7899), anti-SIRT1 (sc-15404), anti-FOS (ABclonal, A0236), anti-SET8 (CST, #2996), anti-UTX (sc-79334), anti-KDM5B (sc-67035), anti-CtBP (Proteintech, 10972-1-AP), anti-MeCP2 (ABclonal, A5694), anti-Nav1.6 (Alomone, ASC-009), anti-H3K9me2 (Abcam, ab1220), anti-H3K9me3 (Abcam, ab8898), anti-G9a (Millipore, 09-071), anti-H3K27me3 (Millipore, 07-449), anti-H3K27ac (Abcam, ab4729), anti-H3K4me1 (Abcam, ab8895), anti-H3K4me3 (Abcam, ab8580), anti-GAPDH and anti-Actin (MBL). A CDYL antibody that we generated (peptides antigen: DGFQGESPEKLDPVDQG and IDDRRDQPFDKRLRFSV; B&M) was used for ChIP-seq, western blotting with rat/mouse cell lysates, and immunohistochemistry. Protein A/G beads were from GE Healthcare Biosciences, protease inhibitor mixture cocktail was from Roche Applied Science. Tetrodotoxin, bicuculline, CGP 55845, and DL-AP5 were from Abcam. Neurobiotin was from Vector Laboratories. Streptavidin Alexa Fluor 488 was from Life Technologies. 4,9-TTX was from Focus Biomolecules. All other reagents were purchased from Sigma-Aldrich.
8
Comprehensive Neurotransmitter Receptor Assays
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All salts with the exceptions of NaCl, KCl, KMeSO4 and PBS were purchased from Sigma Aldrich UK. These were acquired from Fisher Scientific UK. Neurobiotin was obtained from Vector Laboratories UK whilst DL-AP5, bicuculline CNQX, CGP 55845, tetrodotoxin, tetraethylammonium chloride, 4-aminopyridine and nifedipine were purchased from Abcam Ltd (UK). w-agatoxin IVA, w-conotoxin GVIA and SNX482 were acquired from Tebu Bio UK. Alexa 488 steptavidin conjugated antibodies were obtained from Life Technologies UK (now known as Thermo Fisher Scientific UK). TTA-P2 was a generous gift from Merck Laboratories USA.
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