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For the pregnant groups, four placentas and 4 fetuses were also collected from each pregnant mouse, of which half were frozen immediately and the other half were fixed in formalin for further study.
At the end point of the experiment (day 42 for PC3 tumors and day 32 for CL1 tumors), tumors were collected, half were fixed in formalin and embedded in paraffin for H&E staining.
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The first half was fixed in Davison's fixative solution for identification of gonadal developmental stage using a histology method as described below.
The wound samples were divided in half; one half was fixed in McDowell's fixative for 48 h under refrigeration (McDowell and Trump, 1976) for historesin processing for light microscopy and transmission electron microscopy (TEM).
One half was fixed in 10% neutral buffered formalin for histological/immunohistological (IHC) analyses.
One half was fixed immediately, and the other half was cultured in 10% FBS-DMEM/F12 FBS-DMEM/F120 min before fixation.
The posterior region of embryos was bisected and one half was fixed immediately and the other half was cultured for 60 min or 120 min before fixation.
One half was fixed in 4% paraformaldehyde, the other half was used for the measurement of luciferase activity in different brain subregions.
Placentas were separated in two halves, one half was fixed in 1.6% paraformaldehyde with 20% sucrose for further processing and the other half collected for RNA extraction.
The other half was fixed in 10% neutral-buffered formalin and used for immunohistochemical analysis of the Ki67, a parameter of cell proliferation.
The tumors were excised and divided so that one half was fixed in 1% paraformaldehyde and embedded in paraffin, and the other half was flash frozen in OCT compound (Tissue-Tek, Thermo Fisher Scientific).
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