Hi,
Does anyone have some tips for successful freeze cracking? I extract the gonads on a coverslip, fix (leaving approximately 20ul solution), place poly-L-lysine coated slide onto coverslip, and then freeze on a metal plate in dry ice for more than 10 minutes. The problem is that when I remove the coverslip (with a razor) the gonads are coming off with the coverslip. This does not happen every time, but often enough that it is a problem. When it does happen, the solution is coming off in a circular pattern where the gonads are and the rest is sticking to the slide.
I would really appreciate any insight into how to solve this problem.
Thanks,
Toshia
Hi Toshia -
Histobond or Superfrost Plus slides may work better than polylysine-coated slides. Use thin (#1) coverslips (worms tend to stick to the less flexible surface), and for the easiest dissection and freeze-cracking, I suggest siliconizing the coverslips with Surfa-Sil (now called “Hydrocarbon-Soluble Siliconizing Fluid” from Thermo) or equivalent. It’s a pain to do this - you have to dip or drop them into ~1-2%(v/v) Surfa-Sil in chloroform or other solvent, rinse in solvent, then air-dry, but a box will last a long time (you can recover them after cracking them off and re-use them til they break). These racks are crazy-expensive but useful for this purpose: http://goo.gl/GIA3k
Feel free to email me with questions (afdernburg@lbl.gov).
Abby
Thanks Abby. I will give your suggestions a try.
Toshia
have you tried doing dissections with syringe needles?