Can someone help me with some cryosectioning issues?
I made skin tissues in vitro and my protocol is already validated. I've always go for the paraffin cutting but is currently out of service. Then, recently, I've been trying to do frozen sections of this skin. I've never done it before. The skin is presenting some huge holes that is not seen in the parafin-embedded cuts, I think something is wrong with the freezing protocol, as followed:
After the skin is ready, I've placed it on a mold with OCT and dipped it directly in liquid nitrogen for about 10 seconds. Some frozen chucks got so cold that they cracked. After that I left less time completely dipped into liquid nitrogen (5 sec). Then, I've placed the frozen tissue chucks in the cryostat until I proceeded to cut then (10 um sections). After I've placed the sections on a slide, I immediately insert then into formal alcohol for max 40 sec and proceed with normal H&E staining (I know it's ugly tho, but I was testing the procedure).
By the photos I think something is wrong with this process, can someone help me?
This is cryodamage of your tissue caused by incorrect freezing the ice crystals expanded and damaged the tissue. There is a number of issues with your protocol.
1. Fix the tissue first unless your protocol requires an unfixed sample which is a whole other story, but I presume you fix your samples with PFA 2-4% as you mentioned paraffin before.
2. You need to use cryoprotectants such as 15% sucrose incubation until tissue sinks, then move the tissue into 30% sucrose and wait again until it's sinking. Depending on the tissue size may take from a few hours to overnight.
3. Place the sucrose-protected specimens in OCT but don't use liquid nitrogen, liquid nitrogen can't properly freeze samples due to the Leidenfrost effect (the evaporating gas creates the envelope around your sample preventing proper freezing.
4. Instead use Isopentane or Isopropanol as your coolant (any non-toxic liquid with low freezing point will work as well) and add dry ice (preferable) or liquid nitrogen (less preferable due to bubbles) into the coolant. After the coolant liquid cools down (LN2 or dry ice stop boiling to vigorously) use the forceps to hold your OCT mold and touch the bottom of the mold to the liquid.
5. Watch as the mold freezes don't over cool your mold, as long it's freezing and stop being transparent it is sufficient.
6. Store your molds in -20 or -80 in a sealed container as the OCT mold tends to sublimate overtime fast at -20 and slow in -80 thus distorting your sample.
Hope it helps you to get a good morphology. The benefit of cryo is that you don't need an antigen retrieval step and overall have better antibody affinity. Skin is easy tissue to subject to cryosectioning so as long as you follow these steps you should get good histology. One final note never refreeze already sectioned slides as the same cryodamage may occur in the section. If you took the sections from -20/-80 keep them in 4C.
Just to add a few comments, but Anton Lennikov hit the high points.
For small samples/biopsy you don't typically need a cryoprotectant, freezing fast enough of a small size can avoid the ice artifact.
You can use a small reservoir of Isopentane floated in a larger container of LN2 or in a dry ice slurry to avoid the bubbling and cut down on use of reactive reagents.
There are a number of disposable cryomolds that can be used to build a 'block' of OCT media that can be submerged in LN2/Isopentane. This makes it easier to get a regular size, keeps your chucks out of the LN2, and is just easier to hang on to with forceps.
Anton Lennikov it worked well to reduce the holes, but now I'm having trouble to do the hematoxylin eosin staining step, because when I dip the superfrost slide on the water after hematoxylin, the skin gets dragged by water.
Can you help me with the staining protocol too, please?
You can coat your slide glass with Poly-D-lysine that improves bonding. I also found using a hair-dryer simply dry the slides underflow of the cold (not hot!) air. You can find additional methods here if the hair-dryer and Poly-D-lysine doesn't work for you http://www.ihcworld.com/_technical_tips/prevent_section_fall.htm
- The frozen section is known to produce severe artifacts that hinder proper interpretation
- Overfreezing Can cause the section to have holes, but you can solve this problem by polishing the block with a couple of extra turns of the blade to create friction and warm-up the block by pressing on it with your finger (5 - 10 seconds)
Baidaa Abdul - Aziz Mohamed Salah I followed your suggestion and the sections were better. But I still cannot see the cells.
I gess there is atrouble in freezing way . Or ths sections were thick . Which part of skin you deal with ? Did you use the right fixitive ?
Yes, I think the sections were set at 10um. I used Formalin 10% in PBS for fixing before freezing the samples. The skin is just the epidermis that is built in a plastic transwell.
Ater fixint the tissue I rinse in PBS and place tissue in 30% Sucrose. Wait for tissue to drop to the botton of the container. Remove sucrose rinsing ing PBS and place tissue in Tissue Tek cryo mold that contains OCT. Orient skin on edge . I have included a YouTube video that I use to freeze my specimens. "Cryopreservation Techniques". (www.youtube.com/watch?v=MYISj-gCx6g)
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