What is the pore size of the filter frits in the columns for the InsituPro and the mesh size of the baskets for the upgraded InsituPro? The pore size is specified at 35 micron. This is sufficient to process Drosophila and even adult C. Elegans, but not sea urchin embryos or other very small organisms.
For the upgrade, the standard mesh size is 100 µm, but baskets with 55 and 35 µm mesh sizes are available for smaller specimens e. g. sea urchin embryos.
What is the temperature range for incubations?
The thermostated rack can be heated to two freely defined temperatures between ambient and about 80°C. The ambient temperature in the instrument is usually about 25-30°C.
How many reagents can be used?
The reagent rack holds 13 reagents, 1x 175 ml, 5x35 ml and 7x13 ml. Probes are stored in a separate tray.
What if the protocol calls for more than 13 reagents?
Most protocols can be reduced to a standard procedure (Wilkinson Zebrafish and Mouse protocol) not requiring more than 13 reagents. If more reagents are required, a pause can be programmed to exchange reagent solutions by the user.
Can the antibody be cooled?
Two reagent positions (blocking and antibody) are cooled to about 8°C by a peltier cooling element.
Is the antibody incubation performed at 4°C?
No, the antibody incubation has to be performed at ambient temperature as the thermostated rack cannot be cooled. This has been shown to work just as well as incubation at low temperature usually employed in manual protocols.
What are the reagent volumes delivered?
Typical volumes are 150 µl for the small, 350 µl for the medium and 600 µl for the very large columns. As this is considerably less than in manual protocols, most wash steps have to be carried out several times.
Typical volumes for the upgraded InsituPro using baskets is 250 µl for small baskets and 700 µl for medium ones.
Is there any agitation during the incubation steps?
There is no agitation of the specimens in the columns. Agitation is not required due to the design of the incubation columns. The columns are much smaller than the vials typically used in manual protocols and the diffusion distances are relatively short. The diffusion of probes and antibody into the specimens is clearly the rate limiting step, not diffusion across the solution. Diffusion of buffers in wash steps is much faster and here the concentration gradient necessary is built up by frequent solvent exchanges.
For the upgraded InsituPro, a mixing function is included for the basket work areas. If activated, the instrument picks up half of the buffer from each cavity and then dispenses it back. This improves performance for long incubation times.
What is the duration of a typical protocol?
As a rule of thumb, the duration is the sum of all incubation and wash times of a manual protocol. Actual times range from 20 h for Drosophila to 3 days for 13-day-old mice.
Can the specimens be treated with individual or identical probes?
Both are possible. Probes are provided either as individual aliquots or as a common reagent like the antibody solution.
Can the protocol be different between organisms in a single run?
A reasonable variation in incubation times or reagents in some steps of a complex procedure can be programmed. This is done to compare and optimize proteinase K digestion times, for example. However, most of the protocol should be the same for all the specimens in the run.
How is RNAse contamination avoided?
The incubation columns must be sterilized prior to a run. There is also an automated cleaning procedure available for the dispensing needle and tubing. Thorough rinsing procedures are programmed to avoid RNAse contamination even if RNAse digestion has been used as a step in the protocol. The solvent used to rinse the needle is separate from reagents delivered and can be DEPC treated for added protection.
