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As shown in Fig. 4A, the glutamatergic nature of the extracellular fEPSP was confirmed at the end of each experiments through the application of the non-NMDA ionotropic glutamate receptor antagonist DNQX (20 µM), which completely blocked the synaptic "N2 component" without altering the non-synaptic "N1 component" [25].
Calibration of the fluorescent signals was performed at the end of each experiments by adding 10 mM ionomycin in the presence of 3.2 mM Ca2+, to obtain Fmax, followed by 10 mM EGTA (adjusted to pH 8 with 3 mM Tris), to obtain Fmin [50].
The data reported represent the mean ± SD of each experiments performed with 3 replicates tested.
To analyze the differentially expressed mRNA profiles at the end of each experiments, cells were washed with ice cold PBS and stored at −70 °C until RNA was prepared.
In the case of each experiments, the cells were placed in Eppendorf tubes, washed by brief centrifugation with cold PBS, fixed by treatment (20 min in the dark, 4°C) with 250 μl of 4% (w/v) paraformaldehyde (BioLegend, Burlington, ON) and then washed with a mixture of PBS (1 ml) and diluted (PBS) permeabilization buffer (250 μl) containing FBS and saponin (PermWash buffer, BD Pharmingen).
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The length of each experiment was 30 minutes.
The conditions of each experiment are tabulated.
Groups were assigned matched according to body weight at the beginning of each experiment.
That will drive down the cost of each experiment and increase its reliability.
After the conclusion of each experiment, the cold traps were allowed to warm to room temperature.
At the beginning of each experiment, subjects practiced outside the scanner to familiarize with the tasks.
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