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Brain tissues were cut into coronal sections using a cryostat and mounted on glass slides (Superfrost, Fisher, Loughborough, UK) previously coated with poly-L-lysine (Sigma).
The brain tissues were cut equally spaced (thickness 2 mm) coronal blocks, followed by sections sliced into 10 µm in a cryostat.
The paraffin-embedded brain tissues were cut on a cryo-ultramicrotome (Leica, Wetzlar, Germany) into serial 10-μm coronal sections.
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After being immersed in 20% sucrose solution and frozen in liquid nitrogen, coronal sections of the brain tissue were cut into slices of 12 μm in thickness and stored at −20°C for enzymohistochemistry morphological studies.
For immunohistochemical analysis, 3-µm thick sections of paraffin-embedded brain tissue were cut and stained with an antibody to Acanthamoeba spp. (from rabbits immunized with Acanthamoeba genotype T4) at a dilution of 1:2,000.
Parrafin embedded brain tissue was cut at a thickness of 8 µm, and tissue sections were de-paraffinised with xylene and re-hydrated through a stepwise series of ethanol solutions (100 %, 95 %, 70.
Formalin-fixed paraffin-embedded brain tissue was cut into 5 µm sections on a microtome.
Afterwards, brain tissue was cut in a cryostat (−22°C, Microm HM 505 N) into coronal sections at 40 μm thick, which were preserved in a 30% sucrose and 0.1% timerosal in PBS at 4°C [ 26].
Then the brain tissue was cutted into thin blocks, and fixing in 95% ethanol and carrying out the subsequent dehydration and clearing at refrigerator temperatures (4°C).
Flash frozen flank and brain tumor tissues were cut to a thickness of 10 µm.
Four-micrometer-thick sections of brain, skin, bone and heart tissues were cut using a microtome and collected on Superfrost glass slides (Thermo Scientific).
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