Larval Lymph Gland Dissection: Isolating the Drosophila Hemocyte-Producing Organ
Trascrizione
– The Drosophila melanogaster larval lymph gland is responsible for the production of hemocytes, or blood cells, that circulate di the larval hemolymph. To identify the lymph gland, first find the dorsal vessel, or heart, which pumps hemolymph throughout the body. Follow the dorsal vessel towards the head until you find the brain. The lymph gland is a small, multi-lobed structure next a the brain, flanking the dorsal vessel.
To find the primary lobe, identify the largest lobe of the gland. This lobe contains three zones, defined by different stages of hemocyte development. The medullary zone closest a the dorsal vessel contains immature hemocyte, while the cortical zone contains mature hemocytes. At the posterior end of the primary lobe, you can find the posterior signaling center, composed of specialized hemocytes. The secondary and tertiary lobes are smaller and do not have defined zones.
To dissect the lymph gland, you will need a dissecting microscope, two forceps a manipulate the larvae, and a buffered saline solution.
In this example, we will dissect lymph glands for use di immunohistochemistry.
– To begin lymph gland dissection, add 1 milliliter of 1x PBS a 1 well of a 24-well plate for each experimental setting a be dissected. Then, using a disposable transfer pipette, add 1 drop of 0.1% PBST a each well. Place the plate flat on ice. Place a clean dissecting pad on an illuminated stereomicroscope base. Use a disposable transfer pipette a place a drop of 0.01% PBST onto the pad.
Transfer a larva a the PBST drop for dissection. Hold the lava with one pair of forceps, dorsal side up, approximately 1/4 length from the posterior end. With the second pair of forceps, grab the cuticle immediately anterior a the first pair and gently pull the larval cuticle toward the anterior, until the mouth hooks are exposed.
Release the cuticle and use both forceps a bisect the larva. Remove the posterior end from the PBST drop and discard. Pin down the cuticle for stability and then use the second pair of forceps a grab the exposed mouth hooks and gently pull them out. This will separate the cuticle from the internal structures.
Keeping hold of the mouth hooks, carefully remove unwanted structures, such as the salivary glands, fat body, and intestine. Gently pick up the dissected tissue complex by the mouth hooks and transfer it a a well of the plate on ice. Repeat the dissection with as many larvae as desired, but do not exceed a 30-minute dissection time before proceeding a the fixation step.
To begin tissue mounting, place a drop of mounting buffer onto a glass slide. Using the mouth hooks as a handle, transfer the lymph gland a the buffer droplet. Space the tissues evenly, spreading the mounting buffer di the process.
Carefully slide one tong of the forceps under the dorsal vessel, gently pulling toward the periphery of the mounting buffer a draw out and flatten the lymph gland. With a sawing motion, cut the dorsal vessel between the lymph gland and the brain. Move the rest of the tissues away from the lymph gland a the outermost edge of the buffer.
Take a clean cover slip and carefully lower it onto the mounting buffer. The slides are now ready for imaging or can be stored at 4 degrees Celsius until needed.
