Currently microtubule dynamics are observed indirectly through microtubules found in proteins such as EB1, since the alpha, beta tubulin heterodimers are abundant in the cytoplasm. Effective tools for directly tubulin heterodimers are lacking to observe the dynamics of microtubule networks in people. Our protocol utilizes microtubules in neuromuscular synapsis and the muscle cells when combined with the powerful genetic tools available in Enabling or diverse range for therapeutic examinations and genetic screening for the ruler micro tubular network regulatory proteins in the nervous system in people.
Our research focuses on the relationship between the cyto skeletin and the neurodevelopment, as well as its implications for neurological diseases. We hope to discover the mechanism of neurodevelopment and the pathogenesis for neurological diseases by highlighting the impact for cytoskeletal regulatory proteins and neurons. Begin by picking out a wandering drosophila third instar larvae using long blunt forceps.
Wash the larvae with hemo lifelike saline, then dry it with a wipe. Next position the larvae dorsally, or ventrally on a dissection dish under the stereo microscope, depending on the tissue to be observed, opt for dorsal or ventral dissection. Pin the larvas mouth, hooks and tail to maintain extended positioning.
Next, add a drop of the hemo lifelike saline to prevent the larvae from drying. Using dissection scissors, make a small transverse incision close to the posterior end without severing it completely. Proceed to cut along the ventral midline moving toward the anterior end.
Now insert four insect pins each into the corners of the larvae. Make necessary adjustments to the insect pins to maximize the stretching of the larvae. Use forceps to gently remove the internal organs while avoiding harm to the muscles.
To fix the larvae, immerse the carcasses in 100 microliters of 4%paraform aldehyde, while still affixed to the dissecting dish. After this dismount the pins then transfer the sample into a two milliliter micro centrifuge tube filled with 0.2%PBST. Place the tube on a shaker for 10 minutes.
At 15 RPM. Remove the PBST and immerse the paraform aldehyde fixed drosophila, larval carcasses, and a blocking agent for 40 minutes at room temperature. Replace the blocking agent with 200 microliters of the primary antibody diluted with 0.2%PBST.
Leave the larvae to incubate at four degrees Celsius overnight. The next day, wash the larvae with 0.2%PBST for 10 minutes. Next, remove the PBST from the tube and add 200 microliters of diluted secondary antibody.
Incubate it at ambient temperature for one and a half hours in the dark. Introduce a nuclear dye such as TO-PRO(R)3 Iodide into the incubating tube for 30 minutes while staining the microtubules in the dark. Finally, rinse off the secondary antibody and the nuclear dye with 0.2%PBST for 10 minutes.
Begin by placing the antibody labeled drosophila larval carcasses in 0.2%PBST on a glass slide adjusted under the stereo microscope, ensuring that the inner surface of the larval carcass is facing up. Absorb the excess PBST solution using a wipe. Then delicately add a drop of anti-fade mounting medium.
Next, place a cover slip on the slide covering the dissected larvae slowly and gently avoiding bubble formation. Secure the cover slip in place by applying fingernail polish around its edges. Store the slide in a dark location to minimize fluorescent attenuation for image acquisition.
Use the laser scanning confocal microscope with the 63 x oil immersion objective. Then adjust the wavelength and laser power to fit the requirements of the experiment best. To identify the neuromuscular junctions and capture images of muscle four in segment A three.
Select a 488 nanometer laser to activate alpha tubulin or FUCH and 546 nanometers to activate the HRP imaging track, modify the parameters to achieve a frame size of 1024 by 1024 pixels, a digital zoom of 1.0 and an imaging interval of 0.8 micrometers To visualize microtubules in the muscle capture images of muscle two in the segments, a three to a five as it has fewer tracheal branches. Choose a 488 nanometer laser for activating alpha tubulin and a 633 nanometer laser for activating the T3605 imaging track. Adjust the imaging parameters to a frame size of 1024 by 1024 pixels, a digital zoom of 2.0 and an imaging interval of 0.4 micrometers.
Both pre and posts synaptic microtubule organizations of the neuromuscular junction were labeled with anti-alpha tubulin. FUCH staining reflected the abundance of stable microtubules in the pre-synaptic neurons. Decreased signal intensity of anti-FUCH was observed when microtubule severing protein katanin 60 was overexpressed in the presynaptic neurons.
The staining intensity of the axon trunk was stronger than that of the branches. Katanin 60 mutations resulted in increased microtubule loops. Overexpression of Katanin 60 caused short micro tubule fragments within the terminal buttons.
Alpha tubulin staining demonstrated a clear network of microtubules around the nucleus muscle cells had a significantly increased para nuclear micro tubular intensity and exhibited stronger bundles. In the Katanin 60 mutant overexpression resulted in fragmentation of the micro tubule fibers.
