Larval Lymph Gland Dissection: Isolating the Drosophila Hemocyte-Producing Organ

This protocol is an excerpt from Reimels and Pfleger, Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae, J. Vis. Exp. (2016).

1. Larval Lymph Gland Immunohistochemistry

NOTE: The lymph gland is located approximately one-third length from the anterior end of a larva slightly below the brain on the dorsal side. (See arrow in Figure 1B.) The lymph gland flanks the dorsal vessel and is most easily dissected attached to the mouth hooks or to the brain. Wild-type, third instar lymph glands are very small structures; the primary lobes are approximately 100 – 200 µm in length. (See Figure 2A.)

1. To obtain larvae of roughly the same developmental stage for this assay, restrict egg collection by allowing females to lay eggs for a fixed time period of 2 – 6 hr.
2. Lymph gland dissection
   1. For each experimental condition, add 1 ml 1x PBS (Table 1) to one well of a 24-well plate.
   2. Add 1 drop of 0.1% PBST (Table 1) to each well using a disposable transfer pipet.
      NOTE: Detergent, such as Tween 20, is added to lower the surface tension, allowing the tissue to sink to the bottom of the well.
   3. Place the plate flat on ice.
   4. Collect larvae in dissecting dish wells filled with 1x PBS.
   5. Place a clean dissecting pad on an illuminated stereomicroscope base. Use a disposable transfer pipet to place small drops of 0.01% PBST (Table 1) on the pad. Transfer one larva to a PBST drop for dissection.
   6. Hold the larva with one pair of forceps approximately one-quarter length from the posterior end, dorsal side up.
   7. Use another pair of forceps to grab the cuticle immediately anterior to the forceps that are holding the larva. Gently pull the cuticle toward the anterior end until the mouth hooks are exposed.
      NOTE: The aim is to peel the cuticle without disturbing any internal structures.
   8. Release the cuticle and use both forceps to cut the larva in two. Remove the posterior end from the PBST drop.
      NOTE: If the cuticle does not peel all the way to the mouth hooks, cut the larva in two and remove the posterior end. Use one pair of forceps to hold the edge of the cuticle, and use the other pair of forceps to push the mouth hooks through the opening. This will invert the cuticle and expose the mouth hooks. This can be used as an alternate dissection method as well.
   9. Use one pair of forceps to pin down the cuticle (either the ventral cuticle, the dorsal cuticle flap, or both) for stability.
   10. Use another pair of forceps to grab the exposed mouth hooks and gently pull them out.
       NOTE: This will separate the cuticle from the internal structures. Ideally, the eye/antennal imaginal discs, brain, ring gland, and lymph gland flanking the dorsal vessel will remain attached to the mouth hooks.
   11. While still holding the mouth hooks, carefully remove unwanted structures such as the salivary glands, fat body, and intestine.
       NOTE: If the brain separates from the mouth hooks, the lymph gland might still be attached to and collected with the brain. In this case, hold the ventral nerve cord instead of the mouth hooks.
   12. Using the mouth hooks or ventral nerve cord as a handle, transfer the dissected complex containing the lymph gland to the well on ice. Do not exceed 30 min before fixation.
3. Fixation and immunohistochemistry
   1. Place the 24-well plate on the stereomicroscope base.
   2. Use a p200 pipette to carefully remove the PBST from the well.
      NOTE: Empty, neighboring wells are useful to temporarily deposit waste.
   3. Gently add 200 µl 3.7% formaldehyde (Table 1) down the side of the well and swirl the plate to ensure that the dissected tissues are completely submerged.
   4. Return the plate to ice for 30 min. If the lymph glands express fluorescent protein(s), keep the plate covered to prevent photobleaching.
   5. Use a p200 pipette to carefully remove the fixative.
   6. Wash by adding 200 µl 1x PBS to the well and placing the plate on an orbital shaker for 5 min at room temperature. Use a p200 pipette to carefully remove the PBS.
      NOTE: Fixed lymph glands can be left on ice in PBS until lymph glands for all experimental conditions are dissected and fixed.
   7. Repeat the wash steps two more times.
   8. Add 200 µl permeabilization solution (Table 1) to the well. Set the plate on an orbital shaker for 45 min at RT.
      NOTE: 45 min is the "gold standard" for lymph gland permeabilization but the authors have success after only 20 min.
   9. Remove the solution with a p200 pipette.
   10. Dilute primary antibody with antibody solution (Table 1) according to provider's specifications. Add 300 µl primary antibody solution to the well and ensure that the dissected tissues are completely submerged. Incubate at 4 °C overnight.
   11. Use a p200 pipette to remove the primary antibody.
   12. Wash by adding 200 µl 1x PBS to the well and placing the plate on an orbital shaker for 10 min at room temperature. Use a p200 pipette to carefully remove the PBS.
   13. Repeat the wash steps two more times.
   14. Dilute secondary antibody with antibody solution according to provider's specifications. Add 300 µl secondary antibody solution to the well and ensure that the dissected tissues are completely submerged. Incubate on an orbital shaker for at least 2 hr at RT.
   15. Use a p200 pipette to remove the secondary antibody and wash as described in Steps 1.3.12 & 1.3.13. Do not remove the PBS after the final wash.
4. Lymph gland mounting
   1. Clean glass microscope slides using 70% ethanol (Table 1) and tissue wipes.
   2. For each experimental condition, place one drop of mounting buffer (Table 1) on the glass slide.
      NOTE: Two conditions fit on one slide. Mounting buffer volume depends on the number of lymph glands to be mounted. Use 2 µl for approximately 18 – 20 lymph glands, 1 µl for 10 – 12, and 0.5 µl for 5 or less.
   3. Place the microscope slide on an illuminated stereomicroscope base.
   4. For up to two conditions at a time, transfer all of the lymph glands from the well to the mounting buffer with forceps, using the mouth hooks or ventral nerve cord as a handle.
   5. Space the dissected tissues evenly in a circular or rectangular shape, spreading the mounting buffer in the process.
   6. For each of the dissected tissues, individually, slide one tong of the forceps under the dorsal vessel and gently pull toward the periphery of the mounting buffer.
      NOTE: This will draw the lymph gland out from the rest of the dissected tissues and flatten the lymph gland on the glass.
   7. Using one tong of the forceps and a sawing motion, cut the dorsal vessel between the lymph gland and the brain.
   8. Move the rest of the dissected tissues to the opposite side of the lymph gland, at the outermost edge of the buffer.
      NOTE: The unwanted dissected tissues will eventually form a perimeter around the lymph glands and serve as a support upon which the coverslip will rest.
   9. Repeat until all of the lymph glands are separated from the dissected tissues, reserving one of the unwanted dissected tissues to place in the center.
   10. Take a coverslip between two fingers. Check that the coverslip is free of dust and fingerprints. If necessary, use a tissue wipe and 70% ethanol to clean the coverslip.
   11. Ensuring that the edge of the coverslip is parallel to the edge of the glass slide, carefully lower the coverslip over the mounting buffer.
       NOTE: Microscope slides can be stored at 4 °C prior to imaging. Maximum storage time depends on the stability of the antibodies used. 24-well plates can be rinsed and reused.

2. Imaging

1. Image fixed hemocytes and mounted lymph glands on an appropriate standard fluorescence or confocal microscope at 20X or higher magnification according to manufacturer's operating manual. Image whole larvae on a standard stereomicroscope at 2X magnification according to manufacturer's operating manual.
2. Follow software provider's instructions for deconvolution, if desired.