The overall goal of these procedures, is to analyze various components of the Drosophila hematopoietic system with an emphasis on lymph gland dissection and mounting for immunohistochemistry. These methods can help answer key questions in the Drosophila hematopoietic field. Including the effect of genetic or environmental alteration on processes like survival, proliferation, differentiation, and the cell signaling of hematopoietic lineages.
The main advantage of these techniques is that when used in conjunction it can help distinguish specific effects that alterations have on complex processes during hematopoiesis. Begin hemolymph collection by adding 10 microliters of 1x PBS to a microcentrifuge tube and placing the tube on ice. Next place a 10 microliter drop of 1x PBS onto a clean dissecting pad.
Select an individual larvae and dry it by placing it on a tissue wipe then transfer it to the PBS droplet on the dissection pad. Using forceps gently tear open and invert the cuticle to release the hemolymph and then discard the carcass. With the pipette collect the hemolymph from the dissecting pad.
Add the hemolymph to the iced PBS microcentrifuge tube and mix by pipetting up and down. Load 10 microliters of the sample into a hemocytometer chamber and take a concentration reading. To conduct hemolymph immunochemistry first place five microliters of 1x PBS in the center of an 18 millimeter round cover slip situated in the well of a 12 well plate.
Dry a larvae on a tissue wipe and then place it into the PBS on the cover slip. Using forceps gently tear open and invert the cuticle then discard the carcass. Repeat the dissection on separate cover slips with as many larvae as desired and allow hemocytes to adhere to cover slips for five to eight minutes at room temperature.
Do not exceed a 30 minute dissection time before proceeding to the fixation step. Fix the hemocytes by adding five microliters of 7.5%formaldehyde to the cover slips. Let the plate stand for 15 minutes at room temperature.
Wash the cover slips three times with enough 1x PBS to cover the cover slips tipping the plate and aspirating the liquid after each wash. Add 100 microliters of permeablization solution to each well and let stand for 20 minutes at room temperature. Tip the plate and gently tap to aspirate the permeablization solution.
Next add 500 microliters of primary antibody solution to the cover slips and then leave the plates to incubate overnight protected from light if necessary at four degrees celsius. Remove the primary antibody solution and wash the cover slips with PBS for 10 minutes on an orbital shaker setup at low speed. Repeat this wash further two times.
Add 500 microliters of secondary antibody solution to the wells and incubate for at least two hours at room temperature. Remove the secondary antibody solution and wash the cover slips with PBS for 10 minutes with orbital shaking. Repeat this wash step a further two times.
During the wash steps clean a glass microscope slide with 70%ethanol and a tissue wipe. Place five microliters of mounting buffer onto the glass slide for each cover slip paired. Two cover slips can be placed onto a single slide.
Aspirate the PBS from the final wash and then carefully remove the cover slips from the plates. Place the slips inverted onto the mounting buffer on a glass slide. To begin lymph gland dissection add one milliliter of 1x PBS to one well of a 24 well plate for each experimental setting to be dissected.
Then using a disposable transfer pipette add one drop of 0.1%PBS tea to each well. Place the plate flat on ice. Place a clean dissecting pad on an illuminated stereo microscope base.
Use a disposable transfer pipette to place a drop of 0.01%PBS tea onto the pad. Transfer a larvae to the PBS tea drop for dissection. Hold the larvae with one pair of forceps dorsal side up approximately one quarter length from the posterior end.
With the second pair of forceps grab the cuticle immediately anterior to 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 to bisect the larvae. Remove the posterior end from the PBS tea drop and discard.
Pin down the cuticle for stability and then use the second pair of forceps to 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 to the 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 to the fixation step. Transfer the plate to the stereo microscope and use a micro pipette to carefully remove the PBS tea from the well leaving the tissue complexes behind.
Gently add 200 microliters of 3.7%formaldehyde down the side of the well and swirl the plate to submerge the tissue. Return the plate to ice and let stand for 30 minutes. Then transfer the plate back to the stereo microscope and carefully remove the fixative.
Wash the tissue three times by adding 200 microliters of PBS to the well and then placing the plate on an orbital shaker for five minutes at room temperature. Carefully remove the PBS and then add 200 microliters of permeablization solution to the well. Set the plate back onto the orbital shaker for further 45 minutes.
Remove the solution from the tissue and add 300 microliters of primary antibody to the well ensuring the dissected tissues are completely submerged. Incubate at four degrees celsius overnight protected from light if necessary. Remove the antibody solution and add 200 microliters of 1x PBS to the well.
Place the plate onto an orbital shaker for 10 minutes. Carefully remove the PBS and then add 300 microliters of secondary antibodies solution ensuring the dissected tissues are completely submerged. Incubate the tissue on an orbital shaker for at least two hours.
Remove the secondary antibody and wash the tissue three times in 200 microliters of 1x PBS with nutation for 10 minutes. 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 to the buffer droplet.
Space the tissues evenly spreading the mounting buffer in the process. Carefully slide one tong of the forceps under the dorsal vessel gently pulling toward the periphery of the mounting buffer to 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 to 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 four degrees celsius until needed.
This graph shows the average concentration of circulating hemocytes from larvae obtained using the described method. Loss of prothoracicotropic hormone PTTH by genetic ablation of PTTH producing neurons produces a delay in larvae development. Hemocyte concentrations were counted for groups of wild type NPTTH ablated larvae at 120 hours after egg laying and then for the ablated larvae again nine days after egg laying.
The average hemocyte concentration per delayed larvae is significantly less than the control group. Only after nine days after development does the average hemocyte concentration per delayed larvae approach that of the controls at 120 hours. Circulating hemocytes expressing green fluorescent protein are shown here.
The cells were collected, fixed, and incubated with the mixture of plasmatocyte specific antibodies that allow identification of the specific subset of hemocytes. This figure shows a successfully dissected lymph gland of a third instar larvae with primary and secondary lobes flanking the dorsal vessel. Here a larvae lymph gland is seen genetically expressing enhanced blue fluorescent protein in the medillary zone and GFP in the posterior signaling center.
The lymph gland was also immunostained with an antibody against the notch intracellular domain. After watching this video you should have a good understanding of how to prepare various hematopoetic compartments for analysis such as larval lymph glands from immunohistochemistry and circulating hemocytes for concentration measurements or fixation.
