The overall goal of this procedure is to label microtubules in the dendrites of sensory neurons in early stage drosophila larva. This is accomplished by first pinning down a larva in a magnetic dissection chamber, then quickly dissecting it into a larva filet. Then still in the magnetic chamber, the filet is immediately fixed and immunostain.
The final step is to mount the tissue confocal immunofluorescence microscopy can then reveal micro tubial distribution in the dendrites of dopaminergic sensory neuron. Hello, my name is Ol Kareem. Today I will demonstrate a protocol for immuno histological labeling of microtubules in sensory neuro dendrites, trachea, and muscles in the larval body wall.
This method is designed to provide insight into Myco TL organization in the peripheral sensor neurons. In addition, it can be applied to the study of Myco TL networks and other cell types and tissues of the larval body wall Before beginning. It is critical for the success of this protocol that fixative is made fresh immediately before the start of this protocol.
The genetic background of the stock is also critical. For example, to examine dendritic arborization neurons, select animals that express a label in the membranes of those neurons, such as MCDA cherry or MCDA, RA orange. Using a loop of hair quickly wash a first instar larva in water and then move it to the dissection chamber.
Now to examine the ventral cluster neurons, orient the larvae dorsal side up and the ventral side on the glass. To examine dorsal cluster neurons, invert the orientation using an insect pin, secure the anterior end near the mouth hooks. Place the pin close to the end for best results.
Micro, triple networks, and particularly those in sensored DDR, will break down rapidly after the initiation of dissection. Achieving first dissection and quick fixation are key factors in the success of this protocol. Cut the very posterior tip of the larva off with micro scissors using forceps.
Grab the gut tissue poking out from the new opening and gently pull out the whole gut. Now with micro scissors, insert one blade into the same opening and cut along the ventral midline towards the anterior. Now pin the now free posterior corners and gently stretch open the larva filet if required.
Quickly cleaning out the remaining internal organs. Then proceed immediately to fixation. To preserve the fragile microtubules.
Begin by aspirating the calcium free HL 3.1 buffer using a pipette while immediately filling the chamber with freshly prepared fixative. Using a different pipette, immediately replace this diluted fixative. With fresh fixative, All fixation and standing steps are carried out in the dissection chamber.
During this process, be careful not knock the insect pins holding the lava as this MEL to tissue damage. Now allow the fixation to proceed undisturbed for 20 minutes at room temperature to end the fixation. Wash the larva filet clean six times with PBST for 10 minutes per wash.
Thus, the larvas rinsed clean of fixative for a total of one hour at room temperature. Agitation during incubations is unnecessary and is likely to cause inadvertent tissue damage. To prepare for staining, remove the last wash of PBST and block the fixed filet by filling the chamber with 5%Goat serum in PBST, replace the blocking solution with primary antibodies diluted in 5%goat serum In PBST.
The primary antibodies used are mouse, antifa, tubulin, and rat anti CD eight, both diluted one to 1000 to prevent the experiment from drying out. Place the chamber in a plastic container with moist tissue. Incubate the filet in the antibodies for at least 16 hours at four degrees Celsius.
The next day, clear the unbound primary antibodies with six washes of PBST for 10 minutes per wash as was done to remove fixative. After an hour of PBST washes, add the secondary antibody solution diluted in 5%goat serum in PBST. In this example, the antibodies are LOR 4 88, anti mouse IgG and P SI three anti rat IgG cover the chamber from this point forward to prevent Fluor four photobleaching then incubate the filet in the secondary for either two hours at room temperature or overnight at four degrees Celsius, followed by one hour at room temperature.
After the secondary antibody incubation clear the unbound antibodies with the same rigorous schedule of six washes in PBST at 10 minutes per wash. Now the filet is ready for mounting for a quick mount, submerge the tissue in a small drop of 80%glycerol using forceps. Grip the very tip of the anterior end near the mouth hooks and transfer it to a slide cuticle side down seal the sides of the cover slip with nail polish for improved tissue clearing and a permanent stable sample.
Mountain DPX as previously described, fluorescent staining revealed that different branches within a dendrite arbor have different cytoskeletal organization in an arbor of a class four DA neuron from a first instar larvae, the main branches are positive for tubulin. While some thin side branches are not an assemblage of serial sections through a similar staining of a class one DA neuron made into a movie is more revealing when tubulin immuno staining in surrounding tissues makes analysis of micro tubial organization and neurons. Difficult mouse anti-fat tubulin can be substituted with neuron specific mouse antich diluted one to 1000.
The main branches of the class four neurons are fch positive. While some thin side branches are fch negative. An assemblage of serial sections of body wall muscles revealed the microtubule networks in this tissue.
A projection of several confocal sections sectioned through body wall muscles and trachea showed the complex microtubule networks in these tissues. Zooming in an assemblage of serial sections through a tracheal ending in the body wall, muscle revealed the intricacies of the micro tubial organization. So the combination of this procedure with other methods like the generation of larval, peripheral sensor neuron, genetic MXs can answer additional questions such as what mechanisms control micro TL organization in dendrites.
