[Histonet] Desperately seeking help for cryosectioning

[Barbara Schormair]

Hi everybody,

I'm in desperate need of some advice on cryosectioning of whole mouse 
embryos (age E11.5 up to  E14.5). These are fixed in 4% PFA overnight at 
4°C, then kept in 30% Sucrose until they sink to the bottom of the tube 
and then are transfered to molds filled with OTC. For freezing I cool 
down Isopentane on dry ice (15min prior to freezing the embryos), then I 
sink the molds in the isopentane for approx. 30 sec. After that I store 
them at -80°C.
I've used the search option to see earlier postings on this topic, but 
they weren't really useful.
I'm absolutely new to cryosectioning and unfortunately there is noone in 
my group who has any experience on this.
I will give you guys a list of questions that I have (e.g. am I doing 
something wrong) and I would be really happy if someone could at least 
answer some of them. Thanks a lot in advance for your help.

1. Is something wrong with the fixation and freezing procedure, does 
this cause cracks and crumpling of my sections?
2. Is the whole embryo simply too heterogeneous in tissue texture, and 
does that cause cracks and crumpling? If so, should I decalcify or 
better dissect the embryo in e.g. brain, spinal cord and rest of the 
body with inner organs?
3. Which temperature is the best? I've tried from -16°C to -30°C. I also 
don't know how to change the temperature, when my sections start to 
a)crack or crumple b) coil up.
4. How do I prepare the OCT-block for sectioning. Steep or flat edges? 
How much OCT should I leave around the embryo for stabilizing the section?
5. When I start sectioning, the first sections are fine, after 15min 
they start to coil up or crumple, or stick together? Could this be 
caused by increased temperature in the sectioning chamber because it is 
not tightly closed anymore? How far does one close the chamber?

Thanks a lot for any reply, I really need help on this. Are there by 
chance any tutorials or protocols for this available (not only outlining 
the procedures, but explaining why things sometimes don't work, and some 
troubleshooting). Or does someone know a good book on this topic.

Best,

Barbara