Eye Lens Dissection: A Technique to Observe Zebrafish Lens Morphology

프로토콜

1. Dissection of Larval and Adult Zebrafish Lenses

1. Anesthetize fish from 6 dpf larvae to adulthood with tricaine until non-responsive to touch but still showing a strong heartbeat. Measure fish standard length as per Schilling (2002) for staging.
2. Immediately excise eyes using micro-dissection scissors and place into a dissection dish in PBS (Figure 1 shown for adult eyes).
   1. Make a custom 35 mm dish filled with silicone for lens dissections. Once silicone has set, excise a divot around 2-3 mm in diameter and 0.5 mm deep for immobilizing adult eyes/lenses during dissection.
3. Dissection of adult zebrafish lenses
   1. Place an adult eye into the dish divot posterior side up filled with PBS. Immobilize the eye by inserting forceps at <45° angle through the optic disc. Be careful not to nick or compress the lens. Make two or three radial incisions through retina and sclera from the optic disc to the ciliary zone with dissection scissors.
   2. Peel back the retina and sclera like flower petals and invert the eye, cornea side up. Immobilize the lens indirectly via manipulation of the sclera and cornea with the flat side of the scissors, while pulling away the retina and attached tissues with forceps. Carefully trim excess tissue from the lens.
      NOTE: It is vital to dissect carefully to obtain consistently healthy lenses.
4. Dissection of larval zebrafish lenses
   1. Place a larval eye posterior side up onto the flat part of the silicone dish filled with PBS and use a sharpened tungsten needle to make radial cuts through the retina and sclera while immobilizing the eye with another tungsten needle or forceps.
      NOTE: Be careful not to damage the lens.
      1. Sharpen the tip of a 2 cm length of 0.1 mm tungsten wire electrolytically by suspending the wire tip into 10% (w/v) NaOH and applying a low voltage alternating-current14. Secure the needle into a Pasteur pipette by melting the glass end using a Bunsen burner.
         NOTE: Alternative fine dissection tools can also be used.
         CAUTION: NaOH is corrosive.
   2. Gently scoop out the lens from the dissociated eye with a blunt side of the needle, and carefully pull away attached tissue.

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결과

Figure 1: Zebrafish lens dissection. (A) Eyes are dissected from anesthetized fish and placed posterior side up (B). (C) Eyes are immobilized by forceps via the optic disc (od) and two to three radial incisions are made in retina and sclera from the optic disc to the zonules. (D) F...

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자료

Name	Company	Catalog Number	Comments
Phosphate buffered saline (PBS)	Fisher Scientific	BP399
Glass bottom microwell dish (35mm Petri dish, 14 mm microwell, #1.5 coverglass)	MatTek Corporation	P35G-1.5-14-C
Dumont #5 forceps	Dumont & Fils		Keep forceps sharpened
Vannas micro-dissection scissors	Ted Pella Inc	1346	Sharp/sharp straight tips
Confocal microscope	Nikon		Eclipse Ti-E
Sodium Hydroxide beads	Fisher Scientific	S612-3	CAUTION - corrosive/irritating to eyes and skin, target organ - Journal of Visualized Experiments www.jove.com Copyright © 2019 Journal of Visualized Experiments Page 2 of 2 respiratory system, corrosive to metals
Disposable Pasteur glass pipets	Fisherbrand	13-678-20A