Zebrafish भ्रूण आँखों ऊतकों के Microdissection

Summary

इस अनुच्छेद के एक दृष्टिकोण के साथ और बिना रेटिना वर्णक उपकला एक से तीन दिन postfertilization भ्रूण से जुड़ी है, zebrafish retinas microdissect का वर्णन करता है.

Abstract

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 μm, while the diameter of the lens is less 100 μm. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina^{1, 2} and retinal pigment epithelium³.

Protocol

भाग 1: microdissection पहले तैयारी समाधान E3 4 मध्यम (5 मिमी NaCl, .17 मिमी KCl, .33 मिमी 2 CaCl और 0.33 मिमी MgSO 4). घंटी एक समाधान है, 5 (116 मिमी NaCl, 2.9 मिमी KCl, 1.8 मिमी 2 CaCl, 5 मिमी HEPES, pH7.2), फिल्टर निष्फल. 5N NaOH. </l…

Discussion

Zebrafish आँख के ऊतकों के Microdissection प्रभावी ढंग से बरकरार retinas और RPE-संलग्न retinas प्राप्त कर सकते हैं. यह काफी हद तक अभिव्यक्ति एक विशिष्ट नेत्र ऊतक (यानी रेटिना या RPE) से संबंधित अध्ययन में मदद करता है. वास्तव में, हम सफल?…

Materials

Material Name	Tipo	Company	Catalogue Number	Comment
Cordless pestle motor		VWR	47747-370
DC power supply		Lascar	PSU130	Any DC supply would work. The specific voltage of a different machine will need further optimization.
Disposable pestle & microtube, 1.5 mL (DNase, RNase and pyrogen-free)		VWR	47747-366	These are used for tissue collection in TRIzol for expression analysis.
Dumont #5 forceps, Tips: 0.05 x 0.01mm, Inox		World Precision Instruments	500341	Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.
Dumont #5SF forceps, Tips: 0.025 x 0.005mm, Inox		Fine Science Tools	11252-00	Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.
Falcon polystyrene culture plates, 60 X 15 mm		BD Biosciences	351007	These plates are used as dissection plates.
Olympus SZX16 Stereomicroscope		Olympus	SZX16	Any stereomicroscope would work. We used Leica stereomicroscope in previous studies1-3 without any issues. We also use the 1X objective exclusively for the dissection even though we have a 2X objective installed.
Sharpening stone		Fine Science Tools	29008-01	Use this to sharpen the tip of the forceps if necessary
Thermo plate		Tokai Hit	MATS-U55SZX2B	This is used to maintain the temperature of the tissue throughout dissection and minimize the influence of temperature fluctuation on gene expression. We also put the whole microscope in an environmentally controlled room at 28°C during dissection in previous studies1-3 with good success.
Trizol, 100 mL		Invitrogen	15596-026
tungsten wire, 0.015 inch diameter		World Precision Instruments	TGW1510
Wooden Applicator		Puritan	807	This is used for holding the chemically-etched tungsten needle.