Fixation Protocol for Dummies

I am running into 3 major problems with the embryo fixation protocol I am using. Could you please help me to fix them? Or suggest me a better protocol to use?

The protocol I am following is:

  • Embryos on in-house made poly-lysine coated slide (0.1% poly-lysine for the coating);
  • Gently lower coverslip onto slide and let it sit on dry-iced cooled metal block for 4 minutes;
  • Quickly tear off the coverslip using a razor blade and place it in cold (-20) methanol for at least 10 minutes;
  • Wash twice (1XPBS for 5 minutes);
  • Put a drop of mounting media with DAPI (we use vectashield media with DAPI) on a coverslip and flip the slide onto it;
  • Seal borders with clear nail polish.

My problems are:

  1. I lose in general a lot of embryos, with large variation in the loss extent (I generally find ~40 embryos for every 100 embryos I had on slide before fixation. But the range of embryos found can go from 0(!) to 70). Is this large loss normal in your experience? Do you know if there are factors influencing it (e.g.: longer time during the PBS wash)?
  2. I often observe a diffuse background fluorescence on DAPI filter at the mic that makes it very difficult to catch/analyse my embryos.
  3. I find many of my embryos on slide being open, smashed. Presently I am avoiding this by creating room for the embryos with 4 drops of nail polish at the corners of the slide. But I am concerned that this nail polish could in some extent melt during the fixation treatment and ruin the clarity of the slide. Do you know a better method to avoid smashing the embryos?

As a side remark, I fix the embryos with small room available on a bench and as part of a more complicated and time-dependent experiment. This is just to say that ideally the fixation protocol to use should be as easy as possible (few neurons available for it!) to handle.

! Thank you so much for your help !



Here are a few ideas that you may find helpful:

If you are obtaining your embryos with bleaching, make sure that you rinse them well to remove the bleach. Usually 4-5 rinses in 15 mL of M9 works well.

An alternative your your nail polish dots to create room for your embryos is to use ring slides – I have had good luck with Gold Seal Fluorescent Antibody Microslides (you can order them from Fisher). These slides have raised rings on them that prevent the coverslip from smashing the embryos.

When you put the embryos on the slide, let them set 2-5 minutes before putting the coverslip on. It gives the embryos a chance to settle down on the slide and attach to the lysine before the cover slip is added.

After placing the coverslip on the slide, use a Kimwipe to remove as much excess liquid from the slide and the edge of the coverslip. Then when you remove the coverslip, there is less of a chance that the embryos will be embedded in ice and instead will remain attached to the slide.

In my experience, there is still usually some loss of embryos in the course of the freeze cracking, however, hopefully this will reduce some and will also prevent the embryos from being smashed by the coverslip.