This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.

CHAPTER 9 - MOLECULAR METHODS

Sections of Whole Mount in situ Hybridization Preparations

(Source: R. BreMiller)

Resin Sections

1. Use embryos in which alkaline phosphatase has been employed as a detection enzyme. For the best results it is important to overstain 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 of time 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 min each).

4. Replace the methanol with propylene oxide, two changes for 5 min each.

5. Replace with a 1:1 mixture of Epon and propylene oxide for 30 min.

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 min each.

2. Wash in dH2O.

3. Embed embryos in agar-sucrose as described in Agar Mounting, Chapter 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.

4. Wash sections in dH2O 2 to 3 min 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.

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