To stain mutant and control embryos for bacterial beta-galactosidase (LacZ) activity in order to assess the embryonic gene expression of IKMC alleles at embryonic day 12.5 (E12.5).
Description: Whole-mount or sections of stained E12.5 embryos are scored for presence of LacZ staining. Intensity of staining is not required but can be reported. Anatomical terms to localize staining to the structure or substructure will use standard EMAP ontology terms.
Outcrosses (Het x WT) or intercrosses (Het x Het) may be used for the protocol.
For outcrosses, genotype confirmation should be performed if no embryos display positive staining.
For intercrosses, genotype confirmation must be performed on ALL samples to distinguish heterozygous and homozygous stained embryos.
- Embryos are harvested, fixed, processed, and stained according to centre-specific protocols
- Embryos with staining are scored and imaged, concurrent with genotyping to determine zygosity and to identify controls
- Two positively stained heterozygous embryos will be annotated for each mutant line
- At least one heterozygous embryo of each sex (historical non-sexed data is also accepted)
- As annotating homozygous embryos is optional there is no minimal animal or generder requirement.
- Wild type littermate E12.5 embryos that do not exhibit any endogenous mammalian b-galactosidase activity are used as controls. C57BL/6N litters can be stained at each centre’s discretion.
- Embryos are stained in whole-mount according to centre-specific protocols. Centre-specific methods will be provided and be on file and linked to the data for each mutant line
- If there is no staining, all embryos are genotype confirmed.
- When positive staining is found, a representative image will be taken and uploaded for each mutant line. If heterozygous and homozygous embryos are stained, a representative image of each can be uploaded. The image will have a sizing reference bar.
- The metadata collected for embryonic litter will be associated with the image
- Annotations will derive from the heterozygous embryo only.
- For each embryo the scoring system will be: “Expression” (specific staining), “No Expression” (no staining), “Ambiguous” (non-specific, not clear), “imageOnly”. or "tissue not available"
If capturing images please attempt to capture left, right, front, and back views of the embryo.
Quality control for each centre’s processes and annotation will be established by that centre’s staff and published as part of their detailed protocol