This video displays microdissection of retina and retina with retinal pigment, epithelium, or RPE attached from zebrafish embryos at one to three days post fertilization. All procedures can be done with fine forceps and general laboratory supplies. Under standard stereo microscopes for retinal dissection, the single layer RPE is removed and peeled off by brushing action and adherence of the RPE to the surface of the culture plate.
For RPE attached retinal dissection, the adhesive properties of the culture plate are removed by embryonic crush and washing prior to the dissection. To preserve the RPE intact careful lifting of this tissue then efficiently separates the choroid and sclera from the RPE attached retina, followed by lens removal using a chemically etched tungsten needle. This dissection method can obtain intact retina tissues and has been successfully utilized to study tissue specific RNA expression, profiles of zebrafish retina and retinal pigment epithelium.
Hi, I'm Li Jang from the laboratory of Professor UFE Leon in the Department of Biological Sciences at Purdue University. Today we'll show you how to dissect zebrafish embryonic eye tissues. We use this procedure in our laboratory to study the gene network regulating eye development.
So let's get started.Eight. Prior to micro dissection, prepare the following solutions. E three, medium filter sterilized ringer solution and five normal sodium hydroxide.
Next, chemically etch tungsten needles. Set a small beaker in a Petri dish by clay to facilitate handling of the beaker and prevent spillage of sodium hydroxide. Attach a standard paperclip to the side of the beaker.
Pour five normal sodium hydroxide into the beaker until the clip contacts sodium hydroxide. Connect the negative electrode from a DC power supply to the paperclip and the positive electrode to one end of a short piece of tungsten wire. Wrap the other end of tungsten wire with a four millimeter diameter clay ball and dip this end into the sodium hydroxide solution.
Increase the voltage to approximately three volts and dip the wire up and down in one second, intervals in the sodium hydroxide solution for about 10 minutes. The tungsten wire exposed to sodium hydroxide will become gradually thinner when the clay ball drops off the other side of the wire attached to the positive electrode will have a sharp needle shape. Rinse the sharp needle with ringer solution or E three to remove salt deposits and attach the needle to a wooden applicator by tape or a needle holder for easy handling To prepare embryos for dissection.
Maintain adult super fish according to standard protocols the night before breeding. Separate the parent fish in static breeding tanks pairwise using a divider. Remove the divider after the room light is turned on the next morning and allow the parents to cross at 10 minute intervals every half an hour, separating the fish after each crossing.
Maintain the embryos collected from each crossing in E three medium separately at 28 degrees Celsius. Stage embryos at 10 to 12 hours post fertilization and restage immediately before dissection at the specific time points. Use Falcon 60 by 15 millimeter polystyrene culture plates for dissection.
A new unused plate has an intrinsic adhesive property for RPE tissue up to approximately 20 minutes and can aid in the removal of RPE for retinal dissections. If RPE attached retina is desired, this adhesive property can be overcome by crushing two embryos completely in a culture plate containing ringer solution and left for 30 minutes prior to dissection, followed by extensive washing with fresh ringer solution prior to dissection. All fine dissections are done at about five to eight x magnification on an Olympus SC X 16 microscope with a one x objective.
Using dumont forceps or a chemically etched tungsten needle, the microscope stage is warmed to 28 degrees Celsius. To minimize the influence of temperature fluctuation to begin retinal dissection, transfer an embryo at a specific developmental stage to five milliliters of ringer solution in a Falcon 60 by 15 millimeter culture plate prepared as described for retinal dissection without RRPE, cut off the head and part of the anterior trunk using the sharp tip of the forceps quickly removing the zebrafish body Next pin, the posterior end of the head trunk section on a culture plate. With the assisting forceps, open the head on the dorsal side starting from the anterior forehead.
Remove the brain so that the medial side of the eyes is exposed and is facing upward for further manipulations. To dissect the retina, use the top of the forceps to carefully brush the exposed RPE on the medial side of the eye facing upward until a small opening to the retina is seen. Continue the brushing and peeling action until the medial side of the retina is almost all exposed.
Avoid scratching and punching the exposed retina for removal of lateral RPE on the retina of the eye facing the culture dish approach. Had an angle of approximately 45 degrees from the culture plate surface and brush the eye with the forceps. Remove the RPE that is attached relatively firmly to the aura serrata by pinning the detached portion of the RPE to the culture plate for dissection.
And then lift the retina gently to clean up the residual RPE and remove the lens using the adhesive property of the culture dish for adherence. Be careful not to damage the retinal cells, which will also mildly adhere to the dish surface. If the lens does not detach with rolling, use an etched tungsten needle with a swirling action on the lens surface.
To begin RPE attached retinal dissection, use a Falcon 60 by 15 millimeter culture plate prepared as described for an RPE attached retinal dissection. Transfer an embryo at a specific developmental stage to five milliliters of ringer solution. Cut off the head and part of the anterior trunk.
Quickly removing the zebrafish body as done previously. Pin the posterior end of the head trunk section on a culture plate. With the assisting forceps, open the head on the dorsal side starting from the anterior forehead.
Remove the brain so that the medial side of the eyes is exposed and is facing upward. For further manipulations using forceps, lift the eye gently from the posterior lateral side and roll to the anterior side. The choroid and scleral tissue on the outside of the RPE layer are attached relatively tightly to the skin and can be mostly peeled off by rolling the eye carefully to the anterior side.
Remove the lens with the tungsten needle After the lateral side of the eye is exposed and when the eye is still attached to the skin for the retinal dissection. Without RPE and RPE attached retina, the dissected samples can be collected in triol in an RNA re micro tube for downstream. RNA characterization.
A successful retinal dissection without RPE is shown here where the retinal integrity is maintained as indicated by the intact retina surface. The corresponding histological section also shows that the retinal architecture is very well maintained. In particular, the preservation of the extracellular matrix in between the photoreceptor layer and the RPE is exceptionally good.
A successful RPE attached retinal dissection is shown by the intact and continuous RPE layer. The corresponding histological section shows that both retinal and RPE cells are in good shape. The total RNA extracted from the dissected retinas is of high quality.
This is indicated by the electropherogram and gel picture obtained from the bioanalyzer analysis. The baseline is flat and the 18 s and 28 s ribosomal peaks are intact. The RNA integrity number is typically larger than 9.8.
We've just shown you the micro dissection procedures for obtaining zebrafish embryonic retina with and without RPE. When doing this procedure, it's important to remember the most critical steps in retinal dissection are the brushing action by the tip of faucets and the tissue rolling. For RPE remnants removal, we found putting elbows on adjustable arm rest in your chair during dissection can substantially stabilize the hand movements.
So that's it. Thanks for watching and good luck with your experiments.
