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Sectioning X. tropicalis

Sectioning Protocol

Protocol prepared by Roza Vazin and Vivian Choi

Materials:

-       4% low melt agarose (1.6g L.M.P agarose in 40ml of H2O)

-       methanol

-       Ptw (100 ml 10xPBS+1ml Tween-20 to 900ml of H2O)

-       Vibratome (Vibratome Series 1000)

-       Slides (frosted and pre-cleaned, Fisherbrand Cat No. 12-550-34)

-       coverslip

-       Slide warmer

-       Disposable base mold (VWR Scientific Base Molds, 15x15x5 Cat. No 15154-642 )

-       Ted Pella double-edge stainless steel razor blade (Prod No. 121-6)

-       Watchmaker forcep’s (Dumont No.5)

 

Procedure:

Fixation is per usual – 1-2 hours in MEMFA. Then dehydrate into MeOH.

1.) Rehydration (from methanol to Ptw)

5 washes – each for 5 minutes at room temp

q Methanol (MeOH)

q 75% MeOH and 25% H2O

q 50% MeOH and 50% Ptw

q 25% MeOH and 75% Ptw

q 100% Ptw

3x5 minutes in Ptw at room temp

        1.)q

2.)q

3.)q

 

2.) Embedding the embryo in agarose:

-Remove the embryo from the vial of Ptw using a pipet and place it into a base mold.

-Remove as much excess Ptw from the base mold as possible. Then pipet in the melted 4% agarose and fill the base mold up to the very top. The agarose should be melted enough so that it is completely clear and slightly thicker than water.

-Using forceps, position the embryo as desired in the mold. It may be helpful to look at the embryo through a stereoscope at the lowest magnification. Make sure that the embryo is positioned orthogonal to the base of the mold to insure that the sections will be symmetrical. Most commonly, we do coronal sections (sections are made in the dorsal-ventral plane starting from the anterior end and finishing at the posterior end – see figure below). If the embryo is going to be sectioned coronally, it may be best to place the embryo with the head down in the agarose in order to keep it from tilting over. However, this depends on stage, at the late 20’s the embryo balances equally well on its head or tail. Also there is no particular advantage to sectioning from the anterior to the posterior in coronal sections although this is typically how we do it.

-The agarose hardens relatively quickly so it is important to position your embryo in the agarose using forceps soon after adding the agarose.

-It is important to observe the embryo for the first minute as the agarose cools because the position of the embryo may change. It may be helpful to look at the embryo through a stereoscope at the lowest magnification.

 

Various Orientations of the Tadpole:

 

Illustration of tadpole imbedded in agarose and placed on metal block to be cut at coronal orientation:

 

 

 

 

 

3.) Placing the embryo on the mounting block:

-The agarose should harden for at least 20 minutes (it may be longer depending on the initial temperature of the agarose) or until it is firm to the touch and slightly blue. In practice, this can take anywhere from 5-20 min.

-Using a razor blade cut the embryo out of the base mold leaving about 3mm on each side. It is important to leave enough agarose around the embryo so that there is support for the sections. It is also easier to pick up the sections later if the agarose block is larger. However, leaving too much agarose could make sectioning difficult as the agarose might push up against the embryo during sectioning and cause it to tear.

-Glue the embryo onto the metal block using super glue. It is very important that the glue be distributed evenly. It is best to put a dab of glue on the metal block, place the embedded embryo on the block and press down on the embryo to make sure that it is completely level. This is one of the steps of sectioning that may result in the lack of symmetry in the sections. If the glue is not distributed evenly then part of the embryo will be higher than the other, and symmetry will be lost.

 

4.) Setting up the vibratome:

-Cut the razor blade in half at the perforation and place it in the vibratome. Results are best when the razor blade is changed at least every two embryos.

-Add enough water so that the water is level with the edge of the razor so that it touches the blade but does not submerge the blade. This will make sections easier to see and collect. Alternatively, the blade could be submerged. If the blade is not submerged, the agarose block can push against the part of the vibratome holding the blade, which could then cause the section to tear or fall out of the agarose. However, if the blade is submerged, the section floats up in the water and does not push against the blade holder. A larger agarose block can be cut if you submerge the blade in water.

 

            -Place the metal block in the vibratome and tighten the screw.

            -Make sure to lower the block so that the razor blade is slightly higher than

            the top of the gel.

 

5.) Sectioning

-The amplitude and speed should be set depending on how thin the sections are. The vibratome moves the blade in two directions simultaneously: forward through the embryo and laterally. Speed refers to the rate at which the blade moves forward, and amplitude refers to the lateral distance traveled. The thinner the sections, the slower the speed and the higher the amplitude.

-It may take a few attempts to have the agarose positioned directly underneath the razor blade.

-Using a spatula and a thin paintbrush, position the spatula directly below the agarose block, and as a section is made use the brush to sweep the section onto the spatula. Forceps could be used instead of a paintbrush. With forceps, the section can be pinned to the spatula by a corner of the agarose so that it does not slip off of the spatula as you remove it from the water. Sometimes the sections will pop out of the agarose as it is cut, and in this case it is necessary to use a paintbrush to collect the section.

-Transfer the section onto the slide. It is best to add a few drops of water onto the slide before transferring the section. Alternatively, the sections can be stored in an eppendorf tube or vial filled with PTw so that the agarose can be melted off the sections afterwards.

 

6.) Post-sectioning

-Place the slides on the slide warmer to dry them out. It is helpful to have the sections on slides if they are very thin. Certain types of staining, like H&E stains, come out best on sections placed on slides.

-If thicker sections are made (> 100 microns), it is possible to either pop the sections out of their agarose matrix manually using forceps or to melt the agarose off in the water bath. After sectioning, melt the agarose off in a 60 degree water bath. It may be necessary to change the PTw once in order to ensure that the agarose has completely dissolved. Though the time this takes varies according to the size of the agarose block used, it usually falls in the range of 10-15 minutes.

 

Extra Tips:

- Making and maintaining agarose at the correct consistency is one of the most difficult aspects of sectioning tadpoles. If it is not at a correct concentration, sections fall out of the agarose matrix more easily while sectioning which makes them difficult to collect. It may be best to store the initial batch of agarose in 17x60mm, 2DR vials. This prevents the entire batch from being contaminated or undergoing a change in agarose concentration due to water evaporation. Agarose can be kept in a water bath set at 60°C. It is also possible to microwave the agarose before each use but this may cause air bubbles to form.

-It is also imperative that the agarose be hardened to the proper extent, but not dried out, before cutting it out of the base mold. The agarose should be a clear blue and hard to the touch. The agarose should be left to harden for thirty minutes to an hour and a half for optimal results. It is also possible to wrap the base mold up in saran wrap and leave it at 4 degrees for a few hours. Sectioning will not work if the agarose has been left to dry overnight.

-Sectioning at the highest amplitude and lowest speed will result in the cleanest cut. The speed refers to the rate at which the blade moves forward through the specimen. The amplitude is how quickly the blade moves from side to side. It is also important to note that a general rule of thumb is the softer the tissue the slower the forward speed and the higher the amplitude. However, when making sections that are thicker than 100 microns it is not necessary to go as slowly and it is possible to set the speed at 3 or 4. A higher speed can be convenient because it cuts back on the time it takes to section. Speeds may vary among different vibratomes.