Digest at room temperature, with the tube lying on its side (no agitation) (use a 22ºC incubator in high summer or deep winter)
Store PK at -20°c at 10mg/ml – This is 1000x stock
ProteinaseK (PK) digestion times vary with embryo age. Digest according to the table below
Developmental Stage PK Treatment
2 - 10ss quick rinse in 1X PK
36 - 48 hpf 30 - 40 min 1X PK
2.5 dpf 30 - 40 min 1.5X PK
Note: If the embryos are dissected, reduce the incubation time or avoid this step.
3. Post fix in 4% PFA for 20 min at room temperature on gentle shaker
Rinse embryos 3 x 5 mins PBS to remove azide.
Prechill trypsin* solution (0.25% in PBS) and 5mL per tube of PBTr on ice until cold.
Incubate embryos in trypsin in ice for 5-10 mins (according to age 36hpf - 5dpf) may need longer for older embryos, depending on the trypsin batch, titrate upon first use.
Rinse 2x in cold PBTr then 3x10mins in cold PBTr, then bring to RT
*Trypsin stock is 2.5^% (10X)