Getting Those Chromosomes To Spread- A Beginner’s Guide To Meiotic Chromosome Spreads
So you want to do meiotic spreads do you? Maybe it’s to check if meiosis is progressing the way it should, or even to look for sites of DNA damage. Whatever the case this technique can be a little tricky at first, but once you learn a few tricks it’s like riding a bicycle- so grab your bike helmet and lycra shorts and follow us!
A brief intro
Firstly, for those of you who don’t know about the wonderful world of the meiotic spread, here’s a brief introduction. This technique can be used to visualise the pairing, recombination and synapsis of meiotic chromosomes. It can also be used to look at markers of different processes such as DNA repair and DNA damage.
Bursting cells
Chromosome spreads are usually done in conjunction with immunofluorescence to visualize synaptonemal complex structures or distinct foci. The most common problems encountered in this technique are that the cells just don’t burst or that the chromosomes don’t spread enough to distinguish individual foci.
Preparing the slides
The first trick to good chromosome spreads is in the preparation of the slides which should be really clean. So, to begin with, it’s a good idea to wash them overnight in absolute ethanol, then rinse and boil them in dH20 before finally wiping them dry with a lab tissue. It also helps to pre-coat the slides with five drops of 4.5% sucrose solution. The sucrose is hygroscopic, therefore it prevents your preparation from drying out completely. This means that the spreads will last longer and can be stored in the fridge for a few days or even months in the freezer before immunostaining.
When height matters
Once you have your cell suspension (e.g. spermatocytes, oocytes or yeast cells), the cells need to be added to the slide- but not in the normal gentle way- oh no- you’re going to make sure they burst all over the place! In order to help the cells burst, one trick is to drop the cells onto the sucrose-coated slide from a height of about 30 to 40 cm. Some people even take to standing on a chair to get a good ‘dropping height’. This is where you need a good aim and a steady hand otherwise the suspension will end up everywhere (and most likely nowhere near your slide).
Drop, drop, drop…
In addition to the dropping force, two drops of 0.05% TritonX-100 (in dH20) are added to facilitate the bursting process. After 10 min of incubation in the detergent, the spreads are then fixed with eight drops of fixative (2% formaldehyde, 0.02% SDS, pH 8.0) and incubated in a humidified chamber for one hour. The slides should then be briefly dipped a few times in dH20 and air dried before storing at -70ºC until use. As I said before, because of the sucrose solution they can be stored for quite a while before use.
Immunostaining and common markers
If you decide to store your slides in the freezer, simply wash them in PBS for five minutes before starting. The immunostaining part of this technique is pretty much like any other immunofluorescence protocol. It requires a block to prevent non-specific antibody binding, followed by the primary and secondary antibodies.
Use this tip for primary antibodies
Something that seems to work better for the staining is to incubate the spreads with the primary antibody overnight in a humidified chamber at 32?C rather than the usual 4?C and to cover the slides with small pieces of Parafilm to prevent evaporation. Once the cover slips have been mounted with an antifade solution, these slides (if stored at 4?C) can last more than six months with the staining intact.
Common markers
The most common markers used to visualise meiotic progression are SYCP1, 2 and 3 which compose the central and lateral elements of the synaptonemal complex that tethers homologous chromosomes together. Other common markers are MLH2 and 3 for recombination and cross over sites, ?H2AX for double strand breaks and there are many markers for DNA repair (e.g. DMC1 and RAD51).
These are by far just a fraction of what is available to visualize different processes in the spread chromosomes. Whatever you use though, you are sure to get some stunning images if you follow the guide above!