プラスチック製の埋め込みとアフリカツメガエル胚のセクショニング

Hello, my name is Sochi ota. I am a postdoctoral scientist in Dr.Ken Cho's lab at Department of Developmental and Cell Biology in University of California. Irv I Today, I will show you the preparation of simpl section of xenopus embryo.

This procedure includes the bisection of late gas stage frog embryo staining with antibody against the molecule of interest, infiltrate into the plastic and make a five micron same plastic section. This procedure can be utilized to study the immunofluorescence immunohistochemistry or in situ hybridization with very high resolution image. Okay, those are the small handy tools for I use for embryo plastic sectioning of 10 plus embryo.

This is a metal tweezer with delta tip and this one, number one, this can be used to orient the embryo in plastic. And number two, this can be used together with this paint brush to transfer the embryo from microtome to the mounting surface. This is a silicon rubber insulator, which I put on the surface of the slide and which makes a water pull where I can mount the slices of plastic sectioned embryos.

Now I'm showing how to bisect the embryo, fixed embryo for the assay like nstitute hybridization or immunofluorescence detection. And to cut the embryo half, I use this very ultra thin desert grid, which you can buy in grocery store. I think yeah, there are fixed embryo fixing the cell 3.7%formal high overnight.

And that's the, the how you fix it depends on the purpose. This is fixed overnight. For the purpose of incision hybridization, you can go for overnight fixation, but for immunofluorescence detection, which you detect the protein inside of the embryo, I did not recommend too long fixation.

And I rather do the 3.7%form height fixation for only two hours to minimize the influence to the subcellular localization of protein and also avoid the over fixation to allow the nice penetration of the antibody during the detection step. Okay, now I'm dissecting this embryo through the blast wall cutting left side and right side. So now I see that this has stage 11 embryo where the PO is all the way from the dorsal side to the ventral side, but bi a embryo.

And let's take a look at this right hand piece. You see the blast po both in the dorsal side and ventral side, but dorsal side is obvious because this invagination is clearly getting to the deepest deep, deep inside of the embryo compared to the ventral side. So this is a good piece, good bisected piece to study both dorsal and ventral side.

Inside of the embryo, this dissected embryo, I am going to use immunofluorescence detection of protein expressed inside of the embryo. And for immunofluorescence or immunohistochemistry, you use antibody specific antibody for detection. But the problem is antibody doesn't penetrate very well to the inside of the embryo.

So if you use whole mount, whole mount embryo, you cannot know. You cannot study the expression pattern of your proteins at the embryo. But using this pre bisected embryo, you can study what is expression pattern in the cell in deep inside of the embryo.

Ah, stain stained embryo has already been fixed in 3.7%Formaldehyde dehydrated in ethanol and infiltrated into the plastic, which is called techno 7, 100. And this kit contains three part, which is this main part liquid and this powder, which is called hardener one. And this liquid called hardener two.

First I mix this main part and hardener one with 100 to one reio, which is gonna give you this infiltration mix. And that's what is, that is a liquid, which to which I am infiltrated this embryo. And this is still liquid so that it's not gonna harden yet.

And this is those embryo sample are left in this infiltration mix overnights to make sure that embryo sample are completely summarized in this plastic. Now I take this one of the sample embryo with transfer pipette and transfer into thin wall 0.5 MPCL tube, which I use as a embedding mold. Then from this embryo I remove excess liquid.

And now this embryo, this embryo already is ready to be embedded in the hardening plastic. To make the hardening plastic, you mix this infiltration mix with this harden tube at 15 to one. So for example, if I take 750 microliter of this infiltration mix to underpin off tube, then I am supposed to add 50 microliter of this hardener too to initiate the polymerization of the plastic after adding it, oops, close the cap and then mix just gently like this.

And you should not do vortex because oxygen is the inhibitor of polymerization reaction. Add approximately 250 microliter of this mixture to the embryo. And this polymerization step takes almost four hours that you should, you don't have to hurry up at all.

After adding this polymerizing plastic first, you can pipe it up and down to make, make the embryo float in the plastic. And now I take this ed metal tweezer to push around the embryo to orient it so that the cutting surface of the embryo comes to the bottom of this embedding mold. Then close the cap because oxygen is an inhibitor of curing reaction and leave it for four hours to allow ization reaction.

After that, this is a HUD already herd in sample. You put kind of glue to this sample so that to make sure that this plastic hard part is not gonna coming out from this mold. This is the, this is a another plastic which works as a glue for this purpose called techno 30 40.

And I'm mixing this powder and liquid with 2, 2, 1 here is 0.6 gram of this powder. So I am going to add 300 microliters of this liquid, then quickly mix it and this plastic goes becomes hard pretty quickly. So this is a part, you have to be a little bit hard then pour this to this already heard sample.

This is good enough. Close the cap. I look like this.

And this will take approximately 30 to 60 minute to become hard. And this is the one which is ready for actioning. First, I cut the tip of mold with cardboard knife, peel this tip part off from this mold.

So now this embedded embryo are exposed. And also I cut this cap off cause I don't need it. Now I take this tain Roy needle, I mean not needle tain Roy blade, which is harder than the regular stain blade used for parin sectioning.

I briefly dip this into Xin to make it clean. Set this to the, set this to the blade holder and wipe the excess zaine out. And sometime I clean this area with zaine too because cleanness of this area is absolutely important.

Now it's ready for sectioning. Before start sectioning, I set up the mounting chamber. So this is slide glass.

And on top of this I'm putting this silicone rubber in and push farmer push around all the way so that there is not going to, there is not gonna any be any leak. And put this on the slide warmer and pour this clean medice water to make our pool. And put as much as water to make you get until you get the flood surface water surface.

And this is important because this flood water surface gives equal distribution of surface tension of the water, which is important to make the section extend equally. Now I am sectioning, but before doing that I wear this mask because each piece of section is so light, so light and my, my breast can blow away from the stage. So I wear this mask.

And now start sectioning. After you get some number of section piece on the stage, you take this with paint brush and slowly move on top of this mountain chamber and knock this paint brush to make those piece get into the water by gravity. And as soon as they land on the water, they extend to form a circular piece on the surface of the water.

How's that light after overnight dry. Now it's look like this and this. Now the sample is ready to contact in with DPI for nuclear DNA.

And then you can study under the microscope. Today I have shown you the plastic section preparation using late gas stage frog embryo. This procedure is quite useful for to study sub cell or localization of protein, which cannot be studied using paren embedding technique and also this in general for even for residual hybridization.

This plastic section also give you for even for residual hybridization, this procedure can give you much higher level those image than paren or frozen section sample.