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Rehydration (sometimes dehydration is also used, but in reverse order)
Temperature: Ambient
Description: Transfer samples from storage conditions in 100% methanol or ethanol to aqueous buffers through a series of buffer exchanges
bleach (optional)
Temperature: Ambient
Description: Typically incubated with 3% hydrogen peroxide to get rid of internal peroxidase and bleach dark embryos
Alkylation (optional, cannot be done by INTAVIS instrument)
Temperature: Ambient
Description: Acetic anhydride in triethanolamine is used to negatively charge tissue, thereby reducing background
When automated: Acetic anhydride is very unstable in solution, it can be incubated for less than a minute after preparation, so automation is not possible (and probes are generally not required)
cell wall digestion (optional, in plants only)
Temperature: Ambient
Description: Hydrochloric acid is mainly used to hydrolyze cell walls in plant samples
Penetration (time critical)
Temperature: Ambient or 37°C
Description: It is critical to allow the probe to contact the sample tissue
When automated: All proteases should be stable enough not to degrade themselves until use, eg proteinase
hybrid
Temperature: Usually 50-70°C (RNAse at 37°C)
Description: Hybridization consists of several steps:
1.
2.
3.
4.
5.
Antibody Incubation
Temperature: Ambient or lower
describe:
In this step, the antibody binds to the antigen on the probe
1.
2.
3, washing step, washing unbound antibody
When automated: Since essentially all antibodies for detection probes are commercialized, they work at room temperature and have the same specificity
Incubation with alkaline phosphatase buffer (eg NTMT)
Temperature: Ambient
Description: Incubate with freshly prepared buffer in preparation for alkaline phosphatase color reaction
When automated: When using alkaline phosphatase-conjugated antibodies, buffers can precipitate when stored for too long or when prepared prior to use
