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A small percentage of the larvae settled and metamorphosed on the microscopy slides, and those settled individuals were fixed for microscopy (as described below) after attachment.
The brains of all SCA3 patients and control individuals were fixed in a 4% phosphate-buffered, aqueous formaldehyde solution (pH 7.4).
Individuals were fixed in ethanol (95%) and whole DNA was extracted from the mantle using the salting-out method described by [ 121].
T3 seeds from these T2 families were sown on soil and again selected for glufosinate resistance to determine which T2 family individuals were fixed for the FLN1-HA transgene.
In order to prepare Daphnia for transmission electron microscopy (TEM), infected individuals were fixed on ice in 4% glutaraldehyde buffer in Sorensen's phosphate buffer (0.1 M KH2PO4 and 0.1 M Na2HPO4) and kept in the dark for several hours.
All in situ hybridization experiments were performed with multiple specimens (multiple individuals were fixed at regular intervals, within single broods, then repeated at least twice with alternative broods) to fully characterize the expression patterns within and across the three species.
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The number of sampled diploid individuals was fixed to 30 in all populations.
The number of individuals was fixed to n = 10,000 and one single outcome (t = 1) was studied.
If individuals are fixed at lattice sites or direct selection is low, indirect selection result in lineages that structure their local environment, leading to 'smart' individuals and stable patterns of resource dynamics.
This assumed demography, involving periodic bottlenecks where the number of individuals is fixed, is typical of most experimental setups, where daily passages of a sample of the population are performed, and population numbers are experimentally controlled.
HEGs appear to be recurrently gained, degenerate, and lost in a cyclical manner and the intron-HEG combination is eventually lost from a population after all individuals are fixed for these elements [ 9].
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