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After staining with DAPI (1∶10,000), sections were mounted in Mowiol/anti-fade solution (Invitrogen).
Immunostained retinas on slides were mounted in Prolong Anti-fade solution containing DAPI for nuclear staining (Molecular Probes, OR, USA).
After the final wash in TBS, the coverslips were mounted in fluorescence anti-fade solution (Invitrogen) and sealed with clear nail polish to prevent dehydration.
4, 6-dimidino-2-phenylindole (DAPI) (Sigma) was diluted to 2 µg/ml in fluorescence anti-fade solution for nuclear staining and added to coverslips.
After washing, cells were stained with 7.5 ug/ml of rhodamine TRITC-labeled donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories) for 2 h at room temperature and then slides were washed and stained with DAPI and mounted with Anti-Fade solution (Invitrogen) for examination by fluorescent microscopy.
Anti-Fade solution (Vectamount, Vector Labs, USA) was used as mountant.
Slides were counterstained with DAPI and mounted with Prolong anti-fade solution (Molecular probes, Eugene, OR).
Chromosomes were counterstained with propidium iodide or DAPI diluted in Prolong anti-fade solution (Molecular Probes, Eugene, OR).
Afterwards glass slides were washed four more times with PBS, dried and covered with 60 μl anti-fade solution (Citifluor).
The slides were counterstained with DAPI and mounted in an anti-fade solution (Vectashield from Vector laboratories).
Slides were incubated with prolonged anti-fade solution and immunofluorescence images were captured using a Zeiss fluorescence microscope.
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