Objective: Long-term survival monitoring and behavioral characterization of mice across multiple age cohorts (young, mid-age, and old)
Gather these items before starting the experiment. Check off items as you prepare.
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Establish several cohorts of 5 or more mice of both genotypes (Prf1 −/− and C57BL/6 WT controls) for survival and behavioral tests. New cohorts entered the experiment until reaching designated overall cohort size based on previous studies.
Note: Mice assigned to experimental groups according to genotype without randomization or blinding. Equal number of mice from both genders included.
“Several cohorts of 5 or more mice of both genotypes for survival and behavioral tests were established over time and observed for the length of the experiment.”
Observe mice throughout the study period. Exclude any mice that become obese during the first year of experiments from further study.
Note: Mice maintained under specific pathogen-free environment throughout the study
“The mice that become obese during the first year of the experiments were excluded from further study.”
Perform in vivo phenotyping on mice at designated timepoints.
Note: Specific phenotyping methods not detailed in this section
“After in vivo phenotyping, mice were sacrificed at the age of 2 months”
Sacrifice mice at three age timepoints: 2 months (young), 12 months (mid-age), or 24 months (old).
Note: Specific sacrifice method not detailed
“mice were sacrificed at the age of 2 months ('young'), 12 months ('mid age'), or 24 months ('old')”
Collect heparinized blood and plasma from sacrificed mice.
Note: Blood count and biochemistry laboratory was blind to sample identity
“Heparinized blood and plasma were collected. The blood count and biochemistry laboratory was blind to the sample identity.”
Prepare single-cell suspensions from perfused liver, pancreas, lungs, and skin for analysis by flow cytometry and ImageStreamX analysis.
Note: Tissues must be perfused before suspension preparation
“Single-cell suspensions from perfused liver, pancreas, lungs, and skin were taken for analysis by flow cytometry and ImageStream®X analysis.”
Fix tissues in formalin and embed in paraffin blocks for histological analysis.
Note: Alternative to flow cytometry analysis
“tissues were fixed in formalin and embedded in paraffin blocks”
Immediately embed tissues in OCT and store at −80 °C for frozen section analysis.
Note: Alternative to formalin fixation
“tissues were immediately embedded in OCT and kept at −80 °C”
Equal number of mice from both genders included; mice that became obese during first year were excluded; assignment to experimental groups by genotype without randomization or blinding