Mechanical stress assay

Mechanical Stress Assay

To test the impact of added physical stress on the intersegmental vessel (ISV) hemorrhage phenotype observed in collagen mutants.

• To obtain embryos, intercross heterozygous mutant carrier adults of appropriate genotypes (col1a2+/- or col5a1+/- or col1a2+/-; col5a1+/-).
• Raise embryos at 28.5°C. Remove chorion with Pronase treatment at approximately 24 hours post fertilization (hpf).Transfer dechorionated embryosto PTU water to preventpigmentation.
• To begin treatment, separate embryos at 48 hpf into 6-well plates with approximately 30 embryos per well. For the experimental group, replace PTU water with 5 ml of 0.6% methylcellulose solution (diluted in PTU water). Incubate at 28.5°C.
• Embryos are screened approximately every 3 hours during entire assay period for visible hemorrhage and/or hemorrhage-induced scarring in the trunk under standard dissecting microscope.
• For each embryo with visible hemorrhage, the phenotype is documented with the Leica M165 stereomicroscope. Imaged embryos are moved into a separate 24-well plate with appropriate methylcellulose or PTU water solution (one embryo per well).
• Repeat above steps for all embryos until 80 hpf. Embryos are subsequently genotyped individually to correlate the genotype with the phenotype.

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