Hello Margaret
Lets share some experiences with Fiber FISH.
As you probably know here in Leiden the Halo technique was
develloped. This is a good reproducible technique only it is
very laborious. Therefore we tried the Dervish technique but
this turned out to be very reproducible and one of the
biggest disadvantages is that there are a lot of DNA breaks
in the fibers giving rise to strange hyb results. DNA breaks
are due to the force you put on the DNA after lysing the
cells, for Dervish this is gravity, for the Fidlerova method
this is the sliding of the coverslip.
We have develloped a new method which is reproducible but
there is always some variation between the slides. You can
first DAPI stain the slides and look at them defore using.
We use unfixed cells and a very gentle lysis method. We
found out that coated slides are essential, APES gives the
best results, else you lose to much DNA during the
hybridization procedures. Denature the slides only 1-2 min..
Another striking observation is that certain areas on the
slides give the nicest fibers in most cases at the edges, so
try to probe the entire slide.
For your point 2), this is difficult to answer. As we have
quite a lot of experience with FISH it did not really take
long to get things working. Adjusting protocols to our needs
took us a couple of month.
For point 3). Thats were you use it for, to find out new
things.
I allready answered point 4.
If you are interested in the method you can email me.
Hans
