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Tissue sections from the obstructed and the contralateral kidneys of each mouse were fixed in 10% formalin in PBS and embedded in paraffin.
Golgi staining was performed essentially as described previously [17] For electronmicroscopic observation of astrocytes in the CA3 region of the hippocampus, a M5R−/− mouse and a wild-type control mouse were fixed 2.5% glutaraldehyde and 4% paraformaldehyde.
For immunohistochemical staining, lung tissues in C57BL/6 mouse were fixed for at least 24 h by Bouin soultion.
The skin tissues from each mouse were fixed in buffered 10% formalin for 18 h and then embedded in paraffin.
After sacrifice, the back skin and one ear of each mouse were fixed in 10% (v/v) neutral buffered formalin for 24 h at 4°C.
For histological analyses, liver samples (the same lobe from each mouse) were fixed overnight in 10percentneutralral buffered formalin, dehydrated in a graded alcohol series, and embedded in paraffin; 4-µm sections were prepared.
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The resin was allowed to set and the entire mouse was fixed by immersion in 4% buffered formalin.
Dorsal skin of mouse was fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 4 µm.
During microscopy the mouse was fixed with a stereotactic device and maintained at 37°C by use of an appropriately pre-warmed heating mat.
In Figure 1 human and mouse are ' fixed' species and chimpanzee and rat are ' variable' species.
The mouse was fixed on a custom stereotaxic apparatus under a surgical microscope (Leica).
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com