Tissue Preparation for In Situ Hybridization and Immunohistochemistry

1. Dissect embryos in cold L15 media. Rinse with PBS 3 times.
2. Fix for 2 h at 4ºC on nutator in freshly prepared 4% paraformaldehyde/1X PBS. (If embryos are very young, i.e. < St.21 in chick and < E10.5 in mouse, fixation time can be reduced to 1-1.5 h).
3. Rinse 3 times with PBS and wash O/N in PBS at 4ºC on nutator.
4. Sucrose protect for 2 h in 30% sucrose/0.1M PB at 4ºC on nutator.
5. Transfer embryos to plastic chuck and remove extra sucrose with P200.
6. Fill chuck halfway with O.C.T. (Tissue Tek 4583) and arrange embryos under the microscope as desired (usually anterior toward the bottom, dorsal up).
7. Carefully place chuck on top of a piece of dry ice placed on microscope base. Once the bottom has frozen (~30 seconds), transfer chuck to bucket with dry ice to freeze completely.
8. Block can be sectioned immediately or stored at -80ºC indefinitely.

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