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After each sample was rinsed three times using 0.01 M PBS for 5 min, they were incubated with fluorescence-labeled (Alexa Flour) secondary antibodies for 60 min. The slices were mounted on cover slips with an aqueous mounting medium supplemented with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Vector Laboratories Inc., Burlingame, CA, USA).
For most FRET measurements via fluorescence microscopy samples are fixed and mounted on cover slides.
After washing with PBS, the chamber slides were mounted on cover slides with Vectashield mounting medium (Vector Laboratories, Burlingame, USA).
Finally, cells washed three times with PBS were mounted on cover slips in Vectashield mounting medium (Invitrogen) and viewed with a Nikon Eclipse E 600 fluorescent microscope.
Slides were then mounted on cover slips and incubated with anti-NeuN (Neuronal Nuclei, 1∶500), anti-GFAP (GlioFibrilar Acid Protein, 1∶200) or anti-reelin (1∶1000) antibodies for an hour at room temperature.
These structures were mounted on cover glasses.
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They were subsequently washed in phosphate buffer (three times × 40 min), mounted on a cover glass covered with poly-L-lysine (Sigma-Aldrich, St . Louis MO, USA), and embedded in Murray Clear.
In the following, they were washed in PBS (three times x 15 min), mounted on a cover glass covered with poly-L-lysine (Sigma-Aldrich, St . Louis MO, USA), and embedded in Murray Clear or Vectashield (Vector Laboratories Inc., Burlingame, CA, USA).
5 10 embryos were mounted on a cover slip (24×60 mm), desiccated and covered with 10S-Voltalef oil (Lehmann & Voss & Co). as described before (Prokop and Technau, 1993).
For experiments a suspension of 2 µm coated latex beads was added, mounted on a cover slide and sealed.
The nuclei were mounted on a cover slip with low-melting agarose to prevent movement prior to initiation of membrane fusion and confocal imaging.
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