Do You Want to Section Embryos? : The Pros and Cons of Paraffin Sectioning

Before the summer began, I created a stockpile of treated embryos. These embryos, which underwent chemical treatment to impair the cell cycle of the developing neural tissue, were fixed, dehydrated, and stored until needed for further procedures.

The first step in analysis of the treated embryos would be to quantify the difference in cell number between the control and treated embryos. This is vital to the data collection process of this project. With this information, one can prove the hindering effects of the hydroxyurea treatments and their potency based on the concentrations in the treatment groups. In order to count the cells, nucleic acid staining via diamidino-2-phenylindole (more commonly known as DAPI) is used to fluorescently tag the nuclei of the cells in the embryo, which will then be imaged and the cells counted.

To get the embryos to that point, they must be sectioned. This process slices paraffin infused embryos from head to tail end in a sequential manner to be mounted on treated slides. From this, the sections are then released from the paraffin, rehydrated, and stained with DAPI before coverslipping.

Another form of sectioning is through use of the cryostat. In cryosectioning, the embryo is frozen in tissue freezing medium (TFA) and sectioned. The sections are then placed on specially treated slides that are different from the slides used in paraffin sectioning. Because cryosectioning maintains the aqueous nature of the embryos, there is no need to rehydrate the embryo before staining in DAPI.

According to a great majority of people in my lab, paraffin sectioning is much faster than cryosectioning. This is mainly due to the fact that the sections of paraffin embedded embryos come out as a ribbon, whereas the sections in cryosectioning are placed on the slide one by one. This greatly slows down the process. Along those lines, one can easily section 6-12 embryos in an 8 hour day with paraffin sectioning, whereas half the amount of embryos can be sectioned in that same amount of time with cryosectioning.  Also, there is less of a chance of the sections falling off during the cover slipping process which is a common issue with cryosectioning.

Although paraffin sectioning is much faster than its frozen counterpart, it does have some downfalls. Paraffin sections are thinner than cryosections, which makes the imaging process longer and used more slides due to the increased number of sections. Also, the ribbons and paraffin have a tendency to break due to static charge that builds up during the sectioning process.

All in all, the type of sectioning depends on what you plan on getting from the sections. The next step in the process is imaging and counting cells, which will give me a good idea on the inhibitory effects of the treatment and in what area the chemical is targeting in embryonic development.