胚胎和成年斑马鱼眼情的组织学准备Histological Preparation of Embryonic and AdultZebrafish Eyes

INTRODUCTION

Thisprotocol describes the histological preparation of embryonic andadult zebrafish eyes. The methods described here can be easilyadapted for use on other zebrafish tissues.

RELATEDINFORMATION

Thesemethods have been employed in studies on morphological developmentof the zebrafish retina (Branchek and Bremiller 1984, Schmitt andDowling 1999).

MATERIALS

Reagents

0.01 M phosphatebuffered saline (PBS) (pH 7.4) (Sigma)     DPX mountant(Electron Microscopy Sciences, 13512)     Embedding resin forzebrafish eyes   Reagent Amount to add Epon 812 25 mL Araldite 502 20 mL 2-Dodecenylsuccinic acid anhydride (DDSA) 60 mL DMP-30 1.1 mL Solution must be mixed thoroughly. Store at-20°C.     Ethanol (50%, 70%,80%, 90%, and 100%)     Fixation solutionfor zebrafish eyes   1% (w/v)paraformaldehyde (PFA)     3% (w/v)sucrose     2.5% (v/v)glutaraldehyde     0.2 M phosphatebuffer (pH 7.4)   This solution can be stored at 4°C for 2 wk.     Histology stain 1% (w/v) azureB     1% (w/v) methyleneblue     1% osmiumtetroxide     Propylene oxide     Tricaine     Zebrafish embryosor adults of interest

Equipment

Coverslips, No. 1thickness     Embedding mold     Fume hood     Heating block     Knives, glass     Microscope,light     Microtome     Needles (25-30gauge)     Oven, preset to60°C     Razor     Scalpel     Slides, gelatincoated or positively charged (e.g., ESCO Superfrost Plus, ErieScientific)     Spatula, wooden     Stereomicroscope     Transfer pipettes,disposable     Tubes (1.5 mL),microcentrifuge     Tweezers     Vials (15 mL),conical

METHOD

Fixation Collect and fixsurgically removed adult eyes or entire embryonic or larval fish infixation solution:   To dissect and fixadult eyes, euthanize fish in tricaine. Use a scalpel and finetweezers to remove the eyes, and puncture the corneas with a 25- to30-gauge needle. Place the eyes in a 15-mL conical vial with 3-4 mLof fixation solution. Fix at 4°C for 2-3 d. If desired, place oneeye in fixation solution for histology, and process thecontralateral eye for immunohistochemistry in 4% PFA as describedin Immunohistochemistry on Cryosections from Embryonic and AdultZebrafish Eyes (Uribe and Gross 2007).     To fix larval fish,place the entire fish in a 1.5-mL microcentrifuge tube containingfixation solution. Fix overnight at 4°C or at room temperature for4-6 h.     Remove the fixationsolution and wash the fixed samples in 0.01 M PBS:   Wash adult samplesthree times for 15 min per wash.     Wash larval samplesthree times for 5 min per wash.     Fix the samples in1% osmium tetroxide at 4°C:   Fix adult eyes for2-3 h.     Fix larval samplesfor 60-90 min. Avoid light during fixation.     Remove the osmiumtetroxide and wash samples three times in 0.01 M PBS for 5 min perwash.   Osmium tetroxide is toxic and should be disposed of withhazardous waste.     Remove the PBS andproceed with the following dehydration series:   50% ethanol for 5min     70% ethanol for 10min     80% ethanol for 15min     90% ethanol for 20min     100% ethanol for 15min     100% ethanol for 15min     100% propyleneoxide for 10 min     100% propyleneoxide for 10 min     Care should be taken with propylene oxide as it ishighly volatile; propylene oxide incubations and the subsequentsteps should be performed in a hood until the samples are inembedding resin and transferred to an incubator (Step 9).   Thaw embedding resin during the second propylene oxide incubation.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).     Carefully mix equalamounts of embedding resin and 100% propylene oxide in a 15-mLconical tube. Begin resin infiltration with 50% propylene oxide and50% resin for 1 h or longer.   Samples may be left for 5-6 h.     Remove the 50/50embedding resin/propylene oxide mix and add 100% embedding resin tothe samples. Leave the samples overnight at room temperature in thehood with caps open, to allow for propylene oxide evaporation.     The next day,remove the samples and place them in fresh embedding resin in anembedding mold. Under a stereomicroscope, align the samples using asmall needle:   Place one to fiveembryos at the end of a single well, dorsal side up, and align asclosely as possible to each other (Fig. 1A ).     Figure 1. (A) Alignment of 9-dpf (days post-fertilization)embryos in a histology mold. (B) Alignment of an adult eye in ahistology mold.   Place a singleadult eye, lens side up, in a well (Fig. 1B).   It is helpful to place a small piece of paper with an identifyingname or code into each well before adding the resin andsamples.     Bake in a 60°C ovenfor 2-3 d. Baking time depends on the batch of resin. Once theresin has polymerized, the blocks can be stored indefinitely atroom temperature until ready for sectioning.   Sectioning     Trim the block witha razor by cutting excessive plastic (polymerized resin) away fromthe samples. Align the specimen in the microtome block holder toprovide the correct sectioning angle.     Using a glass knifeand microtome, cut thick sections (5-10 µm) until the desiredlocation or depth is reached. To determine location, check thesections often using a light microscope.     Cut semithinhistological sections, 1-1.5-µm thick. Using a wooden spatula (theshaved end of an applicator tip), collect the sections and placethem on a droplet of H2O on a gelatin-coated slide.     Place the slide ona heating block to evaporate the H2O droplet and allowthe specimen to adhere to the slide.     Stain the sectionswith histology stain for 30 sec to 3 min. Stain time varies withthe batch of stain and batch of resin.     Rinse the sectionswith H2O and check the samples on a microscope for thedesired level of staining.   If the sectionshave not absorbed enough stain, repeat Step 14.     If samples areappropriately stained, air-dry for 10-15 min.     Using a disposabletransfer pipette, add three to four small drops of DPXmountant.   The use of disposable pipettes eliminates the risk ofcontaminating more expensive pipettes with the mountant.     Place a No. 1coverslip onto each sample and let the DPX mountant hardenovernight.     Image on a lightmicroscope (Fig. 2 , Fig. 3 ).       Figure 2. Transverse histological section of 5-dpfzebrafish eye stained with histology stain (methylene blue andazure B). The dorsal side is up.     Figure 3. Transverse histological section of an 18-mo-oldzebrafish eye stained with histology stain (methylene blue andazure B). The ventral retina is shown.

ACKNOWLEDGMENTS

This protocoldescribes methods originally developed in John E. Dowling'slaboratory at Harvard University. Work in J.M.G.'s laboratory issupported by the Knights Templar Eye Foundation, the AmericanHealth Assistance Foundation Macular Degeneration Research Program,and the Retina Research Foundation. We thank Polly Harvey fortechnical assistance.

REFERENCES

Branchek, T. andBremiller, R. 1984. The development of photoreceptors in thezebrafish, Brachydanio rerio. I. Structure. J. Comp. Neurol. 224:107-115.   Schmitt, E.A. and Dowling, J.E. 1999. Early retinal development inthe zebrafish, Danio rerio: Light and electron microscopicanalyses. J. Comp. Neurol. 404: 515-536.   Uribe, R.A. and Gross, J.M. 2007. Immunohistochemistry oncryosections from embryonic and adult zebrafish eyes. CSH Protocolsdoi: 10.1101/pdb.prot4779.

Copyright © 2007 by Cold Spring Harbor Laboratory Press.Online ISSN: 1559-6095 Terms of Service All rights reserved. Anyoneusing the procedures outlined in these protocols does so at theirown risk. Cold Spring Harbor Laboratory makes no representations orwarranties with respect to the material set forth in theseprotocols and has no liability in connection with their use.Materials used in these protocols may be considered hazardous andshould be used with caution. For a full listing of cautionsregarding these material, please consult: CSH Protocols; 2007; doi:10.1101/pdb.prot4846 http://www.cshprotocols.org/cgi/content/full/2007/18/pdb.prot4846