Histological preparation of embryonic and adult zebrafish eyes

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1 Histological preparation of embryonic and adult zebrafish eyes Richard J. Nuckels 1 and Jeffrey M. Gross 1,2,3 1 Section of Molecular Cell and Developmental Biology 2 Institute of Cell and Molecular Biology 3 Institute for Neuroscience The University of Texas at Austin 1 University Station, C1000 Austin, TX Corresponding Author: Ph: (512) Fax: (512) jmgross@mail.utexas.edu INTRODUCTION This is a protocol for the histological preparation of embryonic and adult zebrafish eyes (Branchek and Bremiller, 1984; Schmitt and Dowling, 1999). The methods described here can be easily adapted for use on other zebrafish tissues. MATERIALS Reagents 0.2M Phosphate buffer 5.25g sodium phosphate monobasic/h 2 O 23.0g sodium phosphate dibasic/7h Liter H 2 0 Fixation Solution: 1% Paraformaldehyde (PFA) 3% Sucrose 2.5% Glutaraldehyde 0.2M Phosphate buffer ph 7.4 Note: This solution can be stored at 4 C for 2 weeks. Embedding resin: 25 ml Epon ml Araldyte ml DDSA 1.1 ml DMP-30 Must be mixed thoroughly. Store at -20 C.

2 Histology stain 1% Azure Blue 1% Methylene Blue Tricaine 0.01M Phosphate Buffered Saline (PBS), ph 7.4 (Sigma) Osmium tetroxide Ethanol (EtOH) Propylene oxide DPX Mounting Medium (EMS) Equipment Conical Vials, 15mL Coverslips, No. 1 thickness Disposable transfer pipettes Embedding mold Eppendorf Tubes, 1.5mL Fume hood Gelatin coated or positively charged slides (e.g. ESCO Superfrost Plus, Erie Scientific) Glass knives Microtome Oven, 60 C Scalpel Syringes, 15 ml Tweezers Methods Fixation 1. Place surgically removed adult eyes or entire embryonic or larval fish into fixation solution. a. For the dissection and fixation of adult eyes, euthanize the fish in Tricaine. Use a scalpel and fine tweezers to remove the eyes. Puncture the cornea of the eyes with a gauge needle. Place the eyes in a 15 ml conical vial with 3-4ml of fixation solution. Fix at 4 C for 2-3 days. If desired, one eye can be placed in the histology fixation solution and the contralateral eye may be placed into a solution (4%PFA in PBS) for immunohistochemistry and processed as described in Uribe and Gross, b. For the fixation of the larval fish place the entire fish in fixation solution in a 1.5mL eppendorf tube. Fix overnight at 4 C or at room temperature for 4-6 hours. 2. Remove the fixation solution and wash the fixed samples in PBS, 3 times for 5 minutes per wash for embryos, 3 times 15 minutes per wash for adults.

3 3. Fix the samples for minutes in 1% Osmium tetroxide at 4 C. Avoid light. For the adult eyes this fixation time should be extended to 2-3 hours. 4. Remove the osmium tetroxide and wash 3x in PBS for 5 minutes per wash. Osmium tetroxide is toxic and should be disposed of with hazardous waste. 5. Remove PBS and proceed with the following dehydration series: 50% EtOH for 5 70% EtOH for 10 80% EtOH for 15 90% EtOH for % EtOH for % EtOH for 15 Note: The remaining steps should be performed in a hood until the samples are in resin and transferred to an incubator. Care should be taken with propylene oxide as it is highly volatile. Also, the resin is highly toxic until it has polymerized. If possible, devote specific pieces of equipment to resin related work (i.e. 60 C incubator, heating/stirring block, stereomicroscope). 100% Propylene oxide % Propylene oxide 10 (Thaw resin during this incubation step) 6. Carefully mix equal amounts of resin and propylene oxide in a 15mL conical tube. Begin resin infiltration with 50% propylene oxide and 50% resin for 1 hour or longer (can leave for 5-6 hours). 7. Remove the 50/50 resin mix and add 100% resin to the samples. Leave the samples overnight at room temperature in the hood with caps open to allow for propylene oxide evaporation. 8. On the next day remove the sample and place in fresh resin in an embedding mold. Align the samples on a stereomicroscope using a small needle. Place 1-5 embryos at the end of a single well, dorsal side up, and align as closely as possible to each other (Fig. 1A). For adults, place a single eye, lens side up, in a well (Fig 1B). It is helpful to place a small piece of paper with an identifying name or code into each well before adding the resin and samples. 9. Bake in a 60 C oven for 2-3 days (This time depends on the batch of resin). Once the resin has polymerized, the blocks can be stored indefinitely at room temperature until ready for sectioning. Sectioning 10. Trim the block with a razor by cutting excessive plastic (polymerized resin) away from the samples. Align the specimen in the microtome block holder to provide the correct sectioning angle. 11. Using a glass knife and a microtome, cut thick sections (5-10 um) until the desired location or depth is reached. Check the sections often using a light microscope to ascertain the location in the sample. 12. Semi-thin histological sections are cut at 1-1.5um thick. Using a wooden spatula (the shaved end of an applicator tip), collect the sections and place them on a droplet of water on a gelatin-coated slide.

4 13. Place the slide on a heating block to evaporate the water droplet and adhere the specimen to the slide. 14. Stain the sections with histology stain for 30 seconds to 3 minutes. (Stain time varies with the batch of stain and batch of resin). 15. Rinse with water and check the samples on a microscope for a desired level of staining. If the sections have not absorbed enough stain, then repeat step 14. Once the sections are stained to a desired level, air dry then for Add 3-4 small drops of DPX mounting medium using a disposable transfer pipette. (By using a disposable pipette, there is not a risk of contaminating more expensive pipettes with the mounting medium.) 17. Add a no. 1 cover slip and let the mounting medium harden overnight. 18. Image on a light microscope (Fig. 2). ACKNOWLEDGEMENTS: This protocol describes methods originally developed in John E. Dowling s laboratory at Harvard University. Work in J.M.G. s laboratory is supported by the Knights Templar Eye Foundation, the American Health Assistance Foundation Macular Degeneration Research Program and the Retina Research Foundation. We thank Polly Harvey for technical assistance. REFERENCES: Branchek, T and R Bremiller, The development of photoreceptors in the zebrafish, Brachydanio rerio. I. Structure, Journal of Comparative Neurology, Mar 20;224(1): (1984) Schmitt, EA and JE Dowling, Early retinal development in the zebrafish, Danio rerio: light and electron microscopic analyses, Journal of Comparative Neurology, Feb 22;404(4): (1999). Uribe, RA and JM Gross, Immunohistochemistry on Cryosections from Embryonic and Adult Zebrafish Eyes, CSH Protocols: doi: /pdb.prot4779 (2007) FIGURE LEGENDS: Figure 1: (A) Alignment of 9dpf embryos in histology mold. (B). Alignment of adult eye in histology mold. Figure 2: Transverse histological section of 5dpf zebrafish eye stained with methlyene blue and azure blue. Dorsal is up. Figure 3: Transverse histological section of an 18-month old zebrafish eye stained with methlyene blue and azure blue. Ventral retina is shown.

5 Figures: Figure 1A Figure 1B Figure 2

6 Figure 3

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