So I'm working with mouse spinal cords and sometimes the sections are curled/rolled after being sectioned/IHC. It doesn't happen to every batch processed but a good portion do. What would cause that? The tight curling makes it extremely difficult to slide mount. Is it the perfusion? sectioning? iHC protocol? I don't know where to begin troubleshooting.
Here is my current protocol:
The animals are perfused and fixed using 4% para, the lumbar region is removed and placed in a vial of 4% para for one hour. Athens cords are then transferred to a 30% sucrose solution for 3 days then sectioned on a Lecia cryostat at 40micrometers. Sections are placed in cryoprotectant for short term storage for IHCs.
Any assistance would be much appreciated.
Hi Mandy.
I think the curling-effect has something to do with the sharpness of the blade you use for tissue sectioning. You should use a new knife or blade, or an unused spot of the knife for every SC you cut.
The angle of the blade to the specimen is also important. We cut our SCs with an 6° blade angle.
And maybe you should try to cut a bit slower.
Temperature is also important. We cut our SCs at -20°C to -22°C.
But don´t change all parameters at once.
I assume you stain your sections free-floating?
Hope I could help. Please let me know, if some suggestions were helpful.
Sometimes it is a matter of humidiy in the cryo-chamber. But there is no really good way to influence this.
It may help with mounting to use the "backward"-method. Don't cut the whole block through and turn the wheel back. So the section still adheres to the upper edge of the block. Then you can use a brush fo flatten the section on the block-surface and mount it quickly.
In my experience it may be the antirolling acrilic, move back and forth until this does not happen, its position relative to the knife is critical. The angle of the knife is also very important. Vary also the temp, a little bit warmer may be helpful, but you need to play with these variables. Is anybody else using the cryostat? Is he/she moving these things?
I section at -21C and many people use the cryostat (brain, spinal cord, muscle sectioning) so things are constantly being messed with. We do not use disposable blades and my PI doesn't want to sharpen the blade unless it's necessary due to the price and difficulty of finding the grinding medium. I do ignore that and sharpen often but maybe not often enough. Yes, the sections are free-floating.
Thank you all for the suggestions. I'm going to tinker with all you commented on. I'll report back!
First aid is an antiroll plate. If it is adjusted correctly (I do it on OCT (tissue-Tek freezing media) block without tissue which you can keep at-70 for continuous use), it provides for a flat section. You can also warm up the lower cutting edge of the tissue with your finger just before making a section. Try -16 C; it works better for brain tissue. The other approach is embedding in OCT media in a cryomold leaving enough media around the tissue, cut a section and mount on a glass ++ (charged) slide. If the edge of the section curls in this case, it doesn't affect the tissue when you mounting the tissue on a glass slide. If you can use disposable knives (they are not expensive) it will help you as well.
Good luck,
Yelena.
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology