The overall goal of this method is to synthesize amyloid beta 42 peptides and inject them into the adult zebrafish brain, to generate a particular model of Amyloid doses as seen in Alzheimer's disease. This method can help us to address important questions in the neuroscience field, such as the effect of amyloid toxicity on neural stem cells, and how we can elicit the rejuvenation response in such detrimental conditions. The main advantages of this technique are it's report, it's reproducible and the phenotypes we get are highly reminiscent of the Alzheimer's disease in human brains.
The implications of this technique extends towards therapy of Alzheimer's disease because it provides natural vertebrate model, that is capable of activating neural stem cells in disease condition, therefore zebrafish can teach us how to recuperate neurodegeneration. To prepare amyloid beta 42 peptide, load an automated peptide synthesizer with 500 milligrams of FMOC protected resin as the solid phase. Then load 0.5 molar dissolved FMOC protected amino acids in the required volume, as calculated for the respective synthesizer.
Use dimethylformamide of DMF to dissolve the activation HBTU at a concentration of 0.48 molar. Then add 45%volume per volume n methylmorpholine or NMM, the base and 5%volume per volume, acidic anhydride, the capping mixture, in DMF. Load the prepared reagents into the synthesizer and run the automated synthesizer for the solid phase synthesis of the peptide.
After the completion of the peptide synthesis cycles on the synthesizer, cleave the peptides, by transferring the resin to a reaction tube and adding 10 milliliters of a freshly prepared cleavage mixture, consisting of the following. Then continuously mix the solid support in the cleavage mixture for four hours, to cleave the peptides from the solid support. Precipitate the cleaved product by adding the cleavage mixture to 100 milliliters of ice cold diethyl ether.
Pass the solution through a filtration unit containing a PTFE filter with a pore size of 0.45 micrometers. Next, collect the filtered peptide, from the filter paper. And dissolve 100 milligrams of precipitated peptide in 5 milliliters of 1:1 solution of acetonitrile, and distill deionized water, and vortex to dissolve the crude peptide.
Then to purify the peptide, via reverse base HPLC, prepare a semi prepared of HPLC, equipped with a porous polystyrene divinylbenzene column of beat size 10 micrometers. Preheat the column by using a column heating unit, to maintain the column at 50 degrees Celsius. Apply the peptide to the column and collect all the major fractions using an automated fraction collector.
By applying a gradient from 5%to 100%acrylonitrile at four milliliters per minute over 25 minutes. Monitor the chromatogram at 220 nanometers, collect the appropriate peaks and analyze the results using LCMS. The major fractions collected are transferred to sample vials and loaded on to an LCMS machine for an analytical check.
The samples are monitored with a UV detector, while passing the sample through an analytical CA team column of beet size 1.7 micrometer. The correct fractions are identified by mass spectrometry. Lyophilize the correct fractions of the desired peptide to a fluffy powder, in a round bottom flask, by applying a vacuum of 0.052 millibar.
Couple the vacuum pump to a freezing unit, maintained at minus 78 degrees Celsius. Then store the lyophilized powder indefinitely at minus 80 degrees Celsius. Use the lyophilized peptide to prepare a one millimolar stock solution of 1:2:1 mixture of acetonitrile, DMF.
And analytical grade water for the experiments. For injection, prepare a 20 micromolar mixture in PBS. And store the solution on ice until the injection.
Use a needle puller with parameters outlined in the text protocol, to prepare glass injection capillaries. Adjust the pressure setting on the pressure source to 25 PSI. Then on the micro injector, set the hold pressure to 20 PSI, the eject pressure to 10 PSI, the period value to 2.5 and negating value to 100 milliseconds.
Next, load the glass capillary with the injection solution. Then insert the glass capillary into the microinjection holder, and adjust the injection angle to 45 degrees. Place one fish into a new Petri dish, filled with anesthetization solution.
Once it is anesthetized, hold the fish with forceps and orient it for injection. Using the tip of a 30-gauge needle generate a slit in the skull over the optic tectum, where the two lateral plates meet. Use only the tip of the needle and do not penetrate more than one millimeter through the skull.
Then while continuing to hold the fish, insert the tip of the glass capillary through the insertion site. Orient the tip of the glass capillary towards the telencephalon at a 45 degree angle. Then inject one microliter of the solution.
Place the fish back into a transport container until it recovers. Then connect the container to the regularly circulating fish water, to ensure optimum water quality. After sacrificing fish, according to the text protocol, cut open the skull above the optic tectum on the dorsal side using a pointed forcep.
Then use a scalpel to dissect the head just behind the pectoral fin. Place the heads in three milliliters of 2%PFA. And incubate the tissue at 4%Celsius overnight.
For cytoprotection and decalcification, wash the heads thrice in 0.1 molar phosphate buffer, pH 7.4, then transfer them into 20%sucrose, 20%EDTA solution. And incubate at four degree Celsius overnight. Then transfer the heads into 7.5%gelatin, 20%sucrose solution in plastic histology molds.
And freeze them on dry ice. Use a cryostat to section heads into 12 micrometer thick cryosections. Then transfer the cryosections directly on to glass slides and store them at minus 20 degree Celsius for long-term usage.
To stain the slides, dry the sections at room temperature for 30 minutes. After thawing, wash the sections twice in PBS, and once in PBS with 0.3%Triton X-100 called PBSTx. Add primary antibody to the slides.
And incubate the samples at 4%Celsius overnight. The following day, wash the slides once with PBS and twice with PBSTx. Then add to the sections fluorescence coupled secondary antibody along with DAPI and PBSTx.
And incubate the slides in the dark at room temperature for two hours. After three times of washing in PBSTx, use 100 microliters of 70%glycerol and a cover slip to mount each slide. Finally, acquire fluorescent images using a confocal microscope.
To identify the correctly synthesized peptide, mass spectroscopy analysis was performed on all HPLC purified fractions for native amyloid beta 42 and CPP tagged amyloid beta 42 peptides. The HPLC fraction, that yielded one peak on the UPLC, i.e. the correct master charge ratio of the required amyloid beta peptide was further processed for experiments.
After cerebroventricular microinjection, the peptides aggregate in the brain and form amyloid depositions as seen by the green clusters. These aggregates are mostly seen as intracellular depositions, but also around the blood vessels. Such aggregations are indications of a successful accumulation of amyloid peptides and the tissue.
Once mastered, the injection can be performed in few minutes. While attempting this procedure, it's important to closely monitor the health and behavior of the animal. After we developed this technique, it helped us to address questions on Alzheimer's disease, like pathology, and neural stem cell response in the zebrafish brain.
