Tissue Embedding in Sucrose Gelatin and Freezing

This protocol is a modified version of the method proposed by Henrique et al. for avian embryos (12).

1. Immerse tissues in the proper spatial orientation in phosphate buffer-15% sucrose-7% gelatin at 37°C for 1 h, and then let gelatin solidify at 4°C in Petri dishes. A solid block of gelatin-sucrose containing the embryo is formed.

2. Cut out cubes of tissue-containing gelatin and freeze slowly at -60°C in isopentane chilled in liquid N2. Glue the cubes of tissue-containing gelatin onto a peace of cork (2 x 2 cm) using one to two drops of tissue freezing medium (Jung) to maintain correct orientation of the tissue to be sectioned (the anterior part of the embryo will be at the top of the cube and the posterior at the bottom, near the cork). We use a plastic container with 200 to 300 mL of isopentane immersed in liquid N2. Check the temperature with a thermometer; when the isopentane reaches -70°C, remove the container from the liquid N2. Using long forceps, pick up the cork with its attached cube of gelatin. Keep the cube at the surface of the chilled isopentane for about 5 s, and then slowly immerse it into the liquid, in several steps of 5 s each. Once completely immersed, hold the cube in the isopentane for 1 min longer. The cube, now completely white, can be stored for several months at -80°C.

3. Cut 5-^m sections of the embedded tissues on the cryostat at -25 to -28°C. Sections are laid over Super frost slides maintained at room temperature, and can then be stored at -20°C for several months.

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