Sections Of Whole-Mount In Situ Hybridization Preparations
- Jonathan Knight
- Anne Eagle
- ZFIN Admin
(Source: R. BreMiller from Zebrafish Book 5th Edition)
Resin Sections
1. Use embryos in which alkaline phosphatase has been employed as a detection enzyme. For the best results, it is important to over stain heavily the whole-mount preparation.
2. Postfix embryos in 4% paraformaldehyde in PBS. The tissue can be stored in this solution for an extended period in the dark at 4°C.
3. Wash embryos in dH2O and dehydrate quickly through a graded series of methanol (50%, 70%, 95% and twice 100% 2 to 3 minutes each).
4. Replace the methanol with propylene oxide, two changes for 5 minutes each.
5. Replace with a 1:1 mixture of Epon and propylene oxide for 30 minutes.
6. Replace with a 3:1 mixture of Epon:propylene oxide for 2 to 3 hours.
7. Transfer tissue to pure Epon for 4 hours.
8. Embed in fresh Epon. Polymerize overnight in a 60°C oven.
9. Cut sections 5 to 10 μm thick with a glass knife. Dry down on a drop of water. Cover slip in Permount. Alternatively, cover slip in Epon and polymerize at 60°C.
Frozen sections
1. Wash stained embryos in PBS, two changes, 2 to 3 minutes each.
2. Wash in dH2O.
3. Embed embryos in agar sucrose using the method described on page 8.4. Cryoprotect in 30% sucrose and cut 14 to 16 μm sections in a 20°C cryostat. Pick up sections on subbed slides and dry at room temperature 2 to 3 hours.
1. Wash sections in dH2O 2 to 3 minutes and dehydrate through graded alcohols. Clear in xylene and mount in Permount.
NOTES: Aqueous solutions of 0.2% methyl green or 1% neutral red can be used as a counterstain for the frozen sections. Immerse slides briefly (30 to 90 sec) in the staining solution, wash in dH2O, and dehydrate as above.