Protocol - OCT embedding
OCT embedding of tissues/embryos for cryosections
Category:
Histology
Last revision: Mar 08, 2016
Contact
Name: Giovanna L. Liguori
Address: Institute of Genetics and Biophysics Via Pietro Castellino, 111 Naples 80131 ITALY
Email: giovanna.liguori@igb.cnr.it
Figure legend:
Part of Figure 1 from D’Aniello et al., 2013 showing single and double whole mount in situ hybridization (WISH) of Fgf10 and Apj. Fgf10 (blue) marks the secondary heart field (SHF) at E9.5 (I). Apj (red) and Fgf10 (blue) co-localize in the SHF at E9.5 (J). Sagittal sections at E9.5 (J′–J″). h, heart; shf, second heart field. Scale bars: 100 μm (I–J), and 50 μm (J′–J″).
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
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Tissue ready for embedding should be fixed (usually in 4%PFA) and in stored in PBS
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Wash 2 times in PBS 1x rocking
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10 minutes
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Transfer tissue/embryo in Surcrose 10% in PBS
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4° C
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3-4 hours rocking or until the sample drops to bottom of vial
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Sucrose infiltration for CRYOPROTECTION
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Transfer tissue/embryo in Surcrose 20% in PBS
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4° C
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until the sample drops to bottom of vial
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Transfer tissue/embryo in Surcrose 30% in PBS
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4° C
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until the sample drops to bottom of vial
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Transfer tissue/embryo in Surcrose 30% - OCT (1:1) solution
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4° C
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2 hours
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Partially fill dry ice container with dry ice
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Label blocks with each sample number and fill with OTC (TissueTek) freezing compound
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Orient tissue into the bottom of the well and freeze putting on dry ice
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15 minutes
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Place frozen tissue blocks, after they are frozen
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-80° C
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Ready to be sliced after they are frozen completely
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Do not store slides in the cryostat over night, they will dry out
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Other informations
The protocol has been provided by Dr. Giancarlo Bellenchi
Quality validation:
Yes
Validation info
The protocol has been used to produce data published in peer-reviewed journals.
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