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Immunostained sections were then incubated with Hoechst 33342 dye (1 μg/ml, 8 min; Life Technologies) to identify nuclei, rinsed thoroughly, mounted in ProLong Gold anti-fade reagent (Life Technologies), and then air-dried for ≥8 h in the dark.
Dechorionated living embryos at st24 were incubated with 0.5 µM MitoSOX dye for 20 min in the dark, rinsed thoroughly, mounted in low melt agarose and analysed using LSM 700 Zeiss confocal microscope (Carl Zeiss International, Germany).
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Sections were thoroughly washed, mounted, and cover-slipped.
Stained cells were washed thoroughly and mounted with Fluorogel (EMS) and allowed to dry overnight.
After thorough washings, the samples were stained with TO-Pro3-Iodide for nuclear staining for 30 min in the dark, washed thoroughly and mounted with fluorescence mounting medium (Dako).
Sections were then washed thoroughly with PBS, mounted on slides using ProLong Gold (Invitrogen, Carlsbad, CA, USA), and analyzed by confocal microscopy.
The enzyme reaction was revealed with diaminobenzidine (DAB, 0.06%) and H2O2 (0.003%) in 0.01M PBS (40°C) for 15 min. The sections were thoroughly rinsed and mounted on gelatin-coated slides.
Thereafter, sections were thoroughly rinsed and mounted for microscopy.
After being washed thoroughly, cells were mounted and viewed at magnification × 63 with a Zeiss Axioplan imaging microscope, with images being obtained using Zeiss AxioCam at a resolution of 1300 × 1030 pixels.
All stained sections were thoroughly rinsed, dried and mounted with neutral mounting medium (DPX, Klinipath, Belgium).
Slides were washed thoroughly in PBS and mounted in Vectashield anti-fading with DAPI Vector Laboratoriess).
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