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Subsequently, sections were left in a Coplin jar at room temperature for 30 min.
Subsequently, the sections were left in the Coplin jar at room temperature for 20 min.
All sections were left in double distilled H2O at 37 °C for 10 min prior to pepsin (Dako Ref S3002, 1 mg/mL 0.2 M HCl) exposure at 37 °C for 10 min, followed by three rinses in PBS, first of which for 15 min at 27 °C, thereafter at room temperature.
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The middle section is left in an intermediate position for the warmer nine months of the year so that vehicle traffic can use the lower deck of the lift span and pleasure craft can pass under the bridge.
Subsequently, muscle sections were left overnight in a propylene oxide-epon resin mixture in a glass dessicator.
These sections were left overnight in distilled H2O, and the next day they were placed in a first bleaching solution [potassium ferricyanide in potassium chlorate solution, lactic acid] for 60 sec at room temperature.
LR White resin (Electron Microscopy Sciences, Hatfield, PA, USA) was serially added, and the sections were left overnight in 100% LR White.
After heating, the sections were left standing in the antigen retrieval solution for 20 min at room temperature.
Sections were left at 4°C in 25% sucrose in PBS for 24 h to dehydrate.
Fresh sections were left for 5 min in 2 % phloroglucinol in 95%% ethanol and mounted in 6 N HCl.
After four rinses in 0.1 M PBST and two rinses in 0.05 M Tris buffer, sections were left for 1 2 min in a chromagen solution consisting of 0.05% diaminobenzidine (Sigma; Poole, UK), buffer solution and 0.01% H2O2 (DAB substrate kit; Vector Laboratories).
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