Hi, I'm Nikita Ari from the Laboratory of Ralph Boer in the Development and Aging program at the Burnham Institute for Medical Research. I'm Gil ler also from the bottom lab. Today we're gonna show you procedures for the fluorescent labeling of Laval and adult Presolar heart structures.
We use this procedure in the laboratory to prepare the cardiac tubes for morphological and structural analysis via confocal or fluorescent microscopy In order to fluorescently label heart structures in adult drosophila. First, make a single cut between the thorax and abdomen in a semi intact adult to separate body segments. Then trim the overhanging ventral edges of the adult cuticle.
Next, transfer and label adult or dissected larval specimens with primary and secondary antibodies. Following labeling mount washed adult specimens by holding the cuticle edges and placing them heart side down onto a drop of vector shield on a cover slip, invert the cover slip onto a drop of medium flanked by two cover slips on a slide to form a bridge. With the hearts now suspended upright for mounting larval hearts.
Transfer specimens onto a slide with the ventral sides facing down mount two cover slips on either side of the slide. Finally form a bridge with a third cover slip by placing it first on the cover slip near the posterior ends of the larvae. The hearts are now ready for imaging.
Before starting this procedure, prepare the following A DH relaxing buffer containing A DH as described in another video protocol in this series with 10 millimolar EGTA fixative composed of 4%formaldehyde in one X-P-B-S-P-B-S, TX, and appropriately diluted primary antibodies and fluorescently labeled secondary antibodies. In PBS TX dissect adult and larva drosophila to expose the cardiac tubes following the semi intact drosophila heart preparation shown in the video protocols visualizing the beating heart in drosophila and drosophila larval NMJ dissection protocol. But with modifications, use oxygenated a DH during larva dissection and displace the posterior pins slightly from the ventral midline larva.
Cut along the ventral midline. Do not remove any tissue prior to fixation in the following steps. Start the labeling procedure by checking under a microscope that all hearts are rhythmically contracting and relaxing in oxygenated A DH.Then quickly replace a DH with relaxing buffer and reexamine each cardiac tube to ensure contractions are inhibited.
Proceed to fix the hearts by replacing the relaxing buffer with fixative. Incubate at room temperature for 20 minutes with gentle shaking. Note that for larval preparations shaking is not necessary at any step and may even be detrimental to cardiac tissue integrity.
At the end of 20 minutes, remove the fixative and wash the specimens three times for 10 minutes each time with P-B-S-T-X. At room temperature with continual shaking for adults, make a single cut between the abdomen and thorax carefully and cleanly. Separate both body segments to ensure minimal damage to the anterior region of the heart.
Be careful not to cut into the anterior abdominal section where the conical chamber of the heart is. Examine the specimen under the microscope to ensure a clean cut was made. Next, carefully trim back any overhanging ventral edges of the abdominal cuticle, such that what remains is more elliptical and less round.
For larval hearts using fine forceps, carefully remove the fat body. Execute this with extreme caution. Since the larval heart is especially fragile and has very little support from other muscles or connective tissue, do not remove the tracheal branches as this may damage the heart.
Next, incubate with the primary antibody, which is diluted in PVS TX and dispensed at 50 microliters per well of a 96 well plate. For adult flies, hold the trimmed dorsal cuticle by the edges without touching the centrally located cardiac tube and transfer to the plate. Place no more than 12 specimens per well and incubate at room temperature for two hours with continual agitation.
Hold larval specimens by the edge using forceps and transfer to P-B-S-T-X filled. Well again, do not shake the larval specimens at the end of the incubation. Remove the primary antibody solution.
Wash the hearts three times for 10 minutes each time with 100 microliters P-B-S-T-X at room temperature after the last wash at 100 microliters of the secondary antibody in PBS tx supplemented with Alexa 5 9 4 Phin. Keep the samples covered to prevent fluoro four bleaching from now on and incubate for one hour at room temperature. Remove the secondary antibody solution and wash the hearts three times for 10 minutes each time with 100 microliters of P-B-S-T-X at room temperature.
Finally, remove the TRITTON X 100 by rinsing the hearts in 100 microliters of PBS for 10 minutes. The specimens can be stored in the dark at four degrees Celsius for several days before mounting. First, prepare the slide for mounting.
Apply three evenly spaced. 10 microliter drops of vector shield mounting medium to a microscope. Slide place two 18 by 18 millimeter Cover slips on the outer drops, keeping the cover slips about 10 to 15 millimeters apart.
Additionally, place 20 microliters of vector shield in the center of a third cover slip. Next, hold the dissected adult fly by the extreme edges of the cuticle. To carefully remove it from the PVS wash solution, gently place it with the heart side down onto the drop of mounting medium on the third cover slip no more than five hearts per drop on the cover slip.
After placing the hearts on the drop, which is on the cover slip check under a microscope to ensure that all the hearts are facing down. Now carefully invert the cover slip with the hearts and quickly place it on the slide holding the pair of cover slips such that the droplet of the inverted cover slip containing the cardiac tubes fuses with the vector shield droplet resting in the space between the cover slips. Pair in this way, the three cover slips form a bridge.
The hearts will be suspended between the middle cover slip and the microscope. Slide under the bridge check under a microscope to ensure that the adult fly hearts are now facing upward using nail polish. Fix the cover slips at their edges.
The hearts are now ready to be imaged via fluorescent or confocal microscopy. To mount larval hearts, place a drop of about 20 microliters BTA shield on a microscope slide using fine forceps. Grasp the larvae at the edges of the cuticle and carefully transfer it into the drop.
Orient the larvae dorsal side down using tungsten needles. Place up to two larva on one slide. Next, drag each specimen out of the mounting medium and align the larvae in parallel.
Place a cover slip on each side of the aligned hearts using forceps. Place a third cover slip on top first by laying one side on the front cover slip and lowering it down to the rear cover slip. Thus forming a bridge.
Capillary forces will cause a flow of vector shield from rear to front, which helps proper alignment of the larval heart. Finally, fix all the cover slips in place at their edges with nail polish and carefully fill the space between the cover slips with 20 to 30 microliters of vector shield seal with nail polish and store at four degrees Celsius for short term storage or at minus 20 degrees Celsius for a longer period of time. When executed correctly, all components and associated tissues of the dorsal vessel should remain intact and be readily visualized.
The background fluorescence should be minimal for adults. The ventral longitudinal muscle layer stains very well and produces a substantial signal. The underlying circular cardiomyocytes, however, tend not to produce as intense a signal as that which comes from the overlying ventral layer.
The myocytes of the anterior conical chamber of the adult heart contain a substantial amount of contractile material, and consequently, this region appears as the most robust relative to the remainder of the cardiac tube. The image of the larval heart shows a spiraling myo fibrillar arrangement. Similar to that of the adult contractile cardiomyocytes, We've just shown your protocol for preparing and staining ovular hearts for fluorescent and confocal microscopy.
When doing this procedure, it's important to remember to limit any unnecessary handling. This will ensure a better intact and preserved cardiac structure. This protocol only serves as a starting point.
As with any other immunohistochemistry, different antibodies require different conditions and need to be optimized individually. So that's it. Thanks for watching and good luck with your staining.
