Tissue/embryo preparation fo cryosectioning
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| Materials |
|---|
| PBS pH 7.4 |
| 4% PFA in PBS |
| 10%, 20% and 30% sucrose solution in PBS |
| OCT Tissue-Tek |
| Histology moulds |
| Pre-coated glass slides |
| Dry ice |
Procedure:
- Tissues/ anesthetized embryos are dissected and placed into 4% PFA solution overnight at 4° C
- The next day the specimen is washed with PBS and transferred to a 10% sucrose solution for 3 h at 4° C (keep at 4°C until specimen sinks)
- The sample is transferred to a 20% sucrose solution with subsequent incubation in 30% sucrose solution each for 3 hours at 4°C
- Incubate the sample on a rocking platform in a 1:1 mixture of OCT and 30% sucrose for 30 minutes
- Finally the sample is embedded in O.C.T Tissue-Tek using histology moulds, transferred to dry ice and stored at -80°C
- The sections are cut 10 µm thick in a cryostat at -20°C and transferred to pre- coated slides. The temperature of the cutting chamber is adjusted ±5°C according to the tissue specimen
- Sections are allowed to dry at RT for 2 hours and transferred to -20°C for storage