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The skull was opened, and the brain carefully dissected out and placed into 4%% buffered formaldehyde for about 1 week.
After perfusion, the skull was opened and the brain carefully removed and stored for 2 days in a cryoprotective solution (10% sucrose in 0.1 M phosphate buffer, pH 7.4).
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Euthanized animals were perfused with 4%% paraformaldehyde and brains carefully harvested and histopathologically analyzed as described in [ 12].
At 6 or 24 hours post-injection of saline, the mice were removed from their cages, decapitated and their brains carefully removed on ice.
Mice were rapidly decapitated and their brains carefully removed and kept in iced, artificial cerebrospinal fluid (aCSF) with sucrose (in mM sucrose 200, KCl 3, Na2PO4 1.4, MgSO4 3, NaHCO3 26, glucose 10, and CaCl2 0.5).
The entire intact brain was carefully removed and placed on an ice-chilled dish for cleaning.
The brain was carefully removed, halved in the left and right hemisphere and frozen with fine crushed dry ice and stored in a refrigerator at −80°C.
The external appearance of the dead animals, the appearance of the viscera, heart, lungs, stomach, intestine, liver, kidney, spleen and brain were carefully noted and any apparent and significant features or differences from the norm were recorded.
The brain was carefully collected and fixed in paraformaldehyde solution for 24 h followed by 30% sucrose solution for 1 week then covered with optimal cooling temperature (OCT) compound for cryostat sectioning (Ted Pella Inc., Redding, CA, USA) and stored at −80°C until histology was performed.
Following the chronic administration of A. marmelos for 14 days, the vital organs such as heart, liver, kidney, testis, spleen and brain were carefully evaluated by histopathological studies and any apparent and significant changes or differences from the norm were studied.
The cerebellum and the cerebral cortex regions of the brain were carefully dissected, weighed and separately homogenized in 0.1 M phosphate buffer at pH 7.4 with a manual glass-teflon homogenizer on ice to obtain 10% w/v homogenate and centrifuged at 6000 rpm at 4 °C for 25 min to obtain the supernatant, stored in an amber bottle and refrigerated, which was used for biochemical analyses.
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