Cryosectioning Xenopus embryos (Conlon lab)

Protocol submitted by VGP from Conlon lab protocols [1]

link to protocol document [2]

Cryosectioning Xenopus Embryos

Fix embryos to be sectioned for 2 hours in 4% Paraformaldehyde at 4C.

(Note that you should NOT use MEMFA. The embryos will crumble as you try to embed them.)

Rinse 1x in PBS and store in PBS for up to 1 week at 4C.

4% PFA in 0.1 M phosphate buffer

1) heat 60 ml H2O to 65°C

2) add 4 g paraformaldehyde

3) dissolve. If cloudy add a few drops of 6M NaOH

4) @ RT, add 25 ml of 0.4 M phosphate buffer

5) bring to 100 ml w/ H2O

0.4 M phosphate buffer

1) NaH2PO4*H2O 10.6 g (or 9.2 g NaH2PO4)

2) K2HPO4 56 g (or 73 g K2HPO4*3H2O)

3) pH to 7.4 w/ HCl

4) bring to 1 L w/ H2O

Incubate the embryos overnight in 30% sucrose (in PBS) at 4C.

With a plastic Pasteur pipette, place 3-4 embryos on a glass plate. Remove as much sucrose as possible with a Kimwipe, and coat the embryos with OCT freezing medium.

Fill a labeled cryomold (10mm x 10mm x 5mm) with OCT.

Using forceps, gently pick up the embryos one at a time and place them in the mold. Position them so that the are vertical and head down (for transverse sectioning) in the mold, with the head all the way to the bottom. Position them as close together and close to the center of the mold as possible without damaging the tissue.

Snap freeze the mold on dry ice.

Trim the mold around the edges and store at –80C in 50mL conicals. This will keep the blocks from drying out so the tissue does not start to desiccate. The can be stored in this way for up to several months.