Protocol step | Group | ||||
---|---|---|---|---|---|
I | II | III | IV | V | |
Soaking in ddw for 24 h | √ | √ | √ | √ | √ |
Frozen at −80 °C and thawed twice | √ | √ | √ | √ | √ |
Soaking: 75% ethanol Solvent:tissue ratio: 20:1 (v/w) Total duration: 72 h Solvent replacement at 1, 3, 6, 12, 24, 72 h | √ | √ | √ | √ | √ |
Soaking in ddw for 24 h | √ | √ | √ | √ | √ |
Digestion on an orbital shaker using a tissue:enzyme ratio (g/ml) of 1:15 | 0.125% pepsin | 0.125% pepsin | 0.125% pepsin | 0.125% pepsin | 0.125% pepsin |
Solvent | 1× EDTA | 2× EDTA | 1× EDTA | 1× EDTA | 2× EDTA |
Duration (h) | 1.5 | 1 | 2 | 2 | 1.5 |
PBS washing for 30 min; shaking | √ | √ | √ | √ | √ |
DNase and RNase for 6 h; shaking | √ | √ | √ | √ | √ |
Rinsing solution | ddw | ddw | Triton | ddw | Triton |
Duration (h) | 1 | 1 | 2 | 1 | 2 |
Ultraviolet irradiation for 30 min |  |  |  | √ | √ |