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Purpose
To perform a histopathological examination of the brain (striatal, hippocampal and/or cerebellar sections) and to record abnormal findings.
To perform a histopathology examination and annotation on a standard list of parameters of the brain (striatal, hippocampal and/or cerebellar sections).
Experimental Design
Number of mutant animals: 2-3 mutant males
Typical age at experiment 6-16 weeks. Mutants are compared to wildtype mice of the same age
Procedure
- Collect and weigh the brains.
- Drop fix in PFA or 10% Neutral Buffered Formalin and ship weekly.
- Trim the brain producing 3 chunks per brain for the 3 brain areas.
- Place each brain area on its corresponding cassette and incubate at room temperature in 70% alcohol.
- Place cassettes onto tissue processor for 18hr 30min for dehydration with alcohol, replacement with solvent and incubation with paraffin.
- Properly embed the brain chunks in paraffin onto the sectioning cassettes.
- Section each brain area to obtain the 4 critical 5um sections per area to be placed on slides.
- Store the slides in a slide box without a lid to dry overnight at 37oC in an incubator.
- Double stain the slides with nissl-luxol and coverslip.
- Select the best critical section for each area and scan on a Hamamatsu.
- Analyse and record the standard list of parameters for each brain area.
- QC data.
- Deblind, group by genotype and perform statistical analysis.
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Upload and annotate images in Centre-specific database using the standardized IMPC Gross Pathology ontology:
- MA ontology terms for location/topography
- MP ontology terms for large/small/abnormal
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Record all abnormal findings in a Center-specific database using the standardized IMPC Gross Pathology ontology:
- MA ontology terms for location/topography
- MP ontology terms for large/small/abnormal