For which applications is the InsituStain used?
The InsituStain has been designed to perform protocols for in situ hybridization and immunodetection on whole mounts, vibratome sections and slides in a semi-automated fashion. It automates all washing steps in a similar fashion to a manual protocol. However,  individual treatments of specimens may need to be performed outside of the machine. The InsituStain is especially suited for projects not requiring individual treatment of specimens, e.g. mutant screens using a single probe or antibody. It can automate incubation and washing procedures involving steps at elevated or lower temperatures, making it suitable for processing of blots, e.g. Southern and Western. In addition, the used antibody can be recovered to cooled positions.

 

How does the InsituStain work?
The specimens or vibratome sections to be treated are placed in baskets with a mesh at the bottom; slides are placed in a slide holder. Buffers are added to and removed from the incubation tub holding the baskets or slide holders by a peristaltic pump. This allows gentle and fast liquid delivery. The incubation tub holding the samples is placed on a thermostatically controlled shaking platform, allowing incubation of temperatures from 4°C to 80°C whilst shaking.

 

Which steps of the manual protocol are automated?
Typically, all washing steps from rehydration to the stage prior to staining are automated. If incubated with one single probe, even the hybridization step can be automated. Staining is usually performed manually with visual control.

 

How many specimens or slides and how many probes can be processed per run?
The InsituStain is able to process up to 96 small (e.g. zebrafish) specimens or vibratome sections or up to 48 medium-sized (e.g.  mouse embryos) specimens or vibratome sections in parallel. The throughput is lower (up to 24 specimens) for very large specimens requiring large baskets. The slide work area holds up to 20 slides. For special screening projects, a work area for twice the number of specimens is available, but buffer consumption dramatically increases.

If a fully automated protocol is run, only one probe or antibody can be used as all the samples are treated equally. If individual probes are used, the hybridization step must be performed manually.

 

Are the results comparable to those obtained manually?
As the procedure is very similar to a manual protocol, the results are comparable. In addition, the machine allows for improved reproducibility due to standardized protocols.

 

Can the InsituStain be used for a large scale screening project?
The InsituStain is very well suited for a large mutant screen, where all samples are treated uniformly. In this case, the hybridization can be done inside the machine, and the protocol is fully automated. A special work area is available which allows treatment of up to 192 samples in parallel. For a screen using individual probes or treatments, the InsituPro VS is recommended as it can individually treat specimens and use individual probes.

 

Is it possible to recover the antibody or probe?
The InsituStain can recover antibody and probes to cooled positions. All other solutions used can be recovered as well.