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For mice of batch I, blood was collected at sacrifice (PND 49, i.e. three days after boost).
Indeed, increase of DAT-aAbs titers was particularly marked in DAT-i mice of batch I (six-fold over VEH controls).
For mice of batch II, blood was obtained at PND 68, i.e. three weeks after the boost, by lateral tail vein incision (as above).
The experiment was run in two batches of mice (see Experimental Scheme 1): mice of batch I (N = 24) were sacrificed on PND 49, after immunization and boost, to verify the generation of circulating DAT aAbs; mice of batch II (N = 24) underwent a test battery, to evaluate long-term changes in behaviour after immunization and boost.
Mice of batch II were monitored for spontaneous home-cage activity [ 49].
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Plasma levels of DAT aAbs were monitored in mice of both batches, to prove effective immunization.
In this experiment the lowest change of the erythema (local haemoglobin) values was in case of mice batch 2 (where PU_2 was applied) which recommends this sample as a transdermal carrier with the most reduced noxiousness Figures 11 and 12. Figure 11 Melanin evolution ± SEM. Figure 12 Erythema evolution ± SEM.
n denotes the number of different mice, different batches of cremaster muscle or endothelial cells studied in each group.
Five days after the delay-of-reward task, at 3.5 months of age, mice (batch II) were sacrificed; brains were quickly removed, dissected on dry ice and stored at −80°C.
To reduce the bias caused by individual differences between mice and batch variations of CF750-A33scFv-Fc, we further used mice bearing both COLO205 and LS174T tumor grafts to evaluate the uptake of CF750-A33scFv-Fc.
Arterial endothelial cells (ECs) were isolated from the thoracic aortas of C57BL6 male mice (A new batch of cells was isolated from a separate group of three mice for each experiment) and cultured in the EC-specific medium EGM-2 (Lonza, Switzerland) (Yagi et al., 2007) supplemented with 5% foetal serum at 37 °C in a humidified environment with 5% CO2.
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