Part One, Introduction. Hi, my name is Rafael. I'm a postdoc at Professor Stevens Hans Lab at Federal University of Rio de Janeiro in Brazil.
Hi, my name is Ismail. I am a research assistant, also professor. In this video, we'll show how to prepare clear sections of Android bodies.
So let's get started. Part two, materials and equipment. For this procedure, we will need an embedding medium for frozen specimens.
4%Paraform aldehyde solution for fixation of the cells, sucrose solutions for cryo protection. A needle with a slightly bent tip, a stereo microscope for better visualization of the EBS and a shaker. Part three.
Here the human embryo bodies are being cultured in non-adherent culture plates. Using a pipette, we group all the embryo bodies as close together as possible in order to transfer them to a 15 milliliter conical tube. The EBS are carefully harvested and transferred to the tube.
It is important to collect all ebs since there isn't a large amount of material After EBS are decanted, the culture media is carefully discarded. You should be able to see the pellet at the bottom of the tube. Now we can proceed to the fixation procedure.
EBS will be fixed in 4%Paraform aldehyde solution in PBS or phosphate buffered saline. It is important to resuspend the pellet with PFA solution. EBS are then fixed in fixation solution for 30 minutes.
Following fixation, the PFA solution is carefully removed. Taking care to avoid Resus suspending EBS and replaced with a 10%sucrose solution in PBS to initiate cryo protection of the ebs, the material is kept for 30 minutes in this solution. Next, we carefully remove the 10%sucrose solution and resuspend the EBS in a 20%sucrose solution.
Once again, EBS are kept for 30 minutes in this solution. Finally, the 20%sucrose solution is removed and replaced by a 30%sucrose solution in PBS where it is kept for 30 more minutes. Now We can proceed to the orientation and blocking of ebs.
In our lab, we use empty capsule shells as molds. For the blocking of ebs, it is important to coat the internal walls of the tip with sucrose solution before collecting the ebs. Now we can carefully collect the EBS and wait until they decant to the bottom of the tip.
Next, the EBS are placed at the center of the block. This Should be the final position of EBS inside the block. With the help of a stereo microscope and a fine tip, we collect and discharge as much sucrose solution as possible.
Always taking care not to collect the ebs. Using a needle with a slightly bent tip, all EBS are positioned in the center of the block. Finally, the remaining sucrose solution is collected with pieces of filter paper.
The specific location of EBS within the block is very important for obtaining good quality slices. Here is an example of poor EB positioning inside the shell. Following positioning of EBS and removal of sucrose solution, the shell is filled with OCT beginning from the edges, while always taking care to avoid Resus suspending ebs.
The remaining bubbles are removed using a pipette, and the shells Are agitated for 15 minutes. Following agitation. The OCT block is frozen in dry ice.
At This point, you may either store the block wrapped in aluminum foil at minus 80 degrees Celsius for further analysis or proceed to the cryostat Part four, Cutting. To make the cryo sections, you will need a disposable or permanent blade, OCT and poly L lysine pretreated glass slides. The OCT block is removed from the freezer and kept inside the cryostat until it reaches minus 20 degrees celsius.
The block is then positioned on the stand and aligned parallel to the edge of the Blade. EB samples are much smaller than most other tissue samples, so it is very important to be sure that the whole surface of the block touches the blade at the same time, thus minimizing loss of material. After having been positioned, the block is cut into 10 micrometer slices, and these are collected directly onto the glass slides.
The slides may either be stored, preferably at minus 70 degrees Celsius, or may also be used on the same day. The material may be stained for immunohistochemistry or immunofluorescence.Conclusion. The method described herein provides a reasonably inexpensive and easy to follow protocol to obtain thin cryostat sections of embryo bodies developed from both human H nine or mouse US P one stem cell lineages.
The resulting cryo sections may be used in a wide variety of procedures in order to analyze protein expression or EB morphology.
