Whole-Mount Immunofluorescent Labeling of the Mosquito Central Nervous System
- 1Department of Biology, Boston University, Boston, Massachusetts 02215, USA
- 2Center for Systems Neuroscience, Boston University, Boston, Massachusetts 02215, USA
- 3Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA
- 4Center for Neurophotonics, Boston University, Boston, Massachusetts 02215, USA
- ↵5Correspondence: myounger{at}bu.edu
Abstract
Mosquito-borne disease is a major global public health issue. One path toward the development of evidence-based strategies to limit mosquito biting is the study of the mosquito nervous system—in particular, the sensory systems that drive biting behavior. The central nervous system of insects consists of the brain and the ventral nerve cord. Here, we describe a protocol for dissecting, immunofluorescent labeling, and imaging both of these structures in the mosquito. This protocol was optimized for Aedes aegypti and works well on Anopheles gambiae tissue. It has not been tested in other mosquito species, but we anticipate that it would work on a range of mosquitoes, and, if not, our protocol will provide a starting point from which to optimize. Notably, a limited number of antibodies cross-react with Ae. aegypti proteins. This protocol is intended for use with validated antibodies and can also be used to test new antibodies as they are generated. It has been successfully used to visualize protein tags, such as green fluorescent protein, that have been introduced into the mosquito to amplify or detect their presence.
MATERIALS
Reagents
Aluminum foil
Fixative solution for mosquito tissues
Glycerol
Ice
Mosquitoes (male or female adults in a small container [e.g., soup cup covered with insect mesh])
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This protocol is designed for use in Aedes aegypti, although we anticipate the procedure will work across genera.
Mounting medium such as VectaShield (Vector Labs H-1000-10) or SlowFade Diamond (Thermo Fisher S36972)
Nail polish
Permeabilization solution for mosquito tissues
Phosphate-buffered saline (10× without calcium or magnesium [Lonza 17-517Q])
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Dilute 1/10 in double-distilled H2O for 1× PBS.
Primary antibody
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A common antibody that is used to stain the neuropil is anti-NC82 (Developmental Studies Hybridoma Bank [DSHB]) (Wagh et al. 2006) used at a 1/50 dilution. Notably, some Drosophila melanogaster primary antibodies can be used because they have cross-reactivity with mosquito proteins, or an antibody custom-made for mosquitoes can be used.
Secondary antibody
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One commonly used secondary antibody is anti-mouse-488 (Life Technologies A11029) at a 1/500 dilution. Standard secondary antibodies work well, including Life Technologies Alexa Fluor antibodies.
Equipment
Cell strainers
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Prepare these items by cutting the rim off of a cell strainer (Corning 352235) so that it resembles a small basket that can fit inside a well of the 24-well plate. Prepare a basket for each experimental condition.
Confocal microscope and imaging software
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Other microscopes can be used to image the brains as long as they are equipped for fluorescence and are capable of optical sectioning. The imaging parameters will depend on the desired image quality and resolution.
Culture dish (35-mm)
Dissecting stereoscope with light source
Flat-bottom plates (24-well; Corning 353047)
Forceps (very sharp)
Glass Pasteur pipette (long-tip; VWR 14673-043)
Glass slides and coverslips
Ice bucket
Microcentrifuge tube (1.5-mL)
Nutator (set at 4°C)
Orbital shaker (one set at room temperature and another set at 4°C)
Slide holder
Standard freezer box or aluminum foil (see Step 19)
Sylgard 184 silicone elastomer (WPI SYLG184)
METHOD
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Users must follow all institutional procedures for working with mosquitoes and seek guidance from the relevant regulatory bodies for such matters.
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This protocol was modified from Riabinina et al. (2016) and Matthews et al. (2019).
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Perform all dissection steps under a dissecting stereoscope with a light source.
Preparation of Sylgard Plates
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1. A few days before dissection, pour a thin Sylgard slab in a 35-mm culture dish lid according to the manufacturer's instructions.
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This lid is used to dissect because of the shorter lip. The Sylgard slab is used to cushion the head or ventral nerve cord (VNC) and minimize damage to the tissues and the forceps. The Sylgard should be mixed according to the manufacturer's instructions and poured as thin as possible.
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Tissue Removal and Fixation
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2. Anesthetize mosquitoes by placing a small container filled with mosquitoes on ice in an ice bucket. It will take ∼5 min until they are anesthetized.
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3. Once the mosquitoes are anesthetized, perform the following steps under a dissecting stereoscope to begin isolating tissues.
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i. Place the mosquito on the 35-mm culture dish lid containing a Sylgard slab.
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For Brain Dissection
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ii. Carefully remove the heads of adult mosquitoes from the body by pinching at the neck with sharp forceps.
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iii. Quickly place heads in a 1.5-mL tube containing 1.25 mL of fixative solution on ice. Between one and 30 heads can be placed in a tube, depending on the experimental needs.
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For Ventral Nerve Cord Dissection
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iv. Carefully remove the body of the adult mosquito from the head by pinching at the neck with sharp forceps.
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v. Quickly place the entire body (without the head) in a 1.5-mL tube containing 1.25 mL of fixative solution on ice. Between one and five bodies can be placed in a tube, depending on the experimental needs.
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4. Place the samples on a nutator for 3 h at 4°C.
Dissection and Blocking
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5. Prepare a 24-well plate by adding a single cell strainer and 0.5 mL of wash solution to each well in the first column of the 24-well plate.
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Multiple samples can be processed together in one cell strainer, but different genotypes or conditions should be kept in separate strainers and labeled for identification.
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6. Perform dissections as follows under a dissecting stereoscope.
For Brain Dissection
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i. Fill the 35-mm culture dish lid containing a Sylgard slab with ice-cold PBS and transfer a single head to the dish with forceps.
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ii. Dissect the brain out of the head capsule in ice-cold PBS with very sharp forceps.
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Take care not to puncture the brain and to delicately remove the esophagus.
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iii. Transfer the dissected brain to a cell strainer containing wash solution in the 24-well plate by using a long glass Pasteur pipette.
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Be careful to only suction the brain into the first inch of the pipette, otherwise it may get stuck to the side and lost.
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For Ventral Nerve Cord Dissection
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iv. Fill the 35-mm culture dish lid containing a Sylgard slab with ice-cold PBS and transfer a single body to the dish with forceps.
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v. Dissect the VNC out of the thorax in ice-cold PBS with very sharp forceps.
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Take care not to puncture the tissue as you delicately remove the surrounding musculature.
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vi. Transfer the dissected VNC (which will resemble a translucent gummy bear) to a cell strainer containing wash solution in the 24-well plate by using a long glass Pasteur pipette.
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Be careful to suction the VNC only into the first inch of the pipette, otherwise it may get stuck to the side and lost.
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7. Wash the tissue in wash solution for 15 min at room temperature by placing the plate on the orbital shaker set to a low speed.
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8. For each strainer in use, fill the next available well in the plate with 0.5 mL of wash solution and transfer the cell strainer containing the tissue to the well containing the fresh wash solution. Wash the sample for 15 min at room temperature by placing the plate on the orbital shaker.
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9. Repeat Step 8 for six total washes.
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10. Transfer the cell strainer containing tissues to a well containing 0.5 mL of permeabilization solution and place the plate on an orbital shaker for 48 h at 4°C.
Primary Antibody Incubation
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11. Add 0.5 mL of wash solution to a clean well and wash the tissue for 15 min at room temperature by placing the plate on the orbital shaker.
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12. Transfer the cell strainer containing the tissue to the next well in the plate that contains fresh wash solution. Carry out six total 15-min washes in fresh wash solution at room temperature on the orbital shaker.
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13. Dilute primary antibodies in antibody buffer in a 1.5-mL microcentrifuge tube on ice and mix by pipetting or inverting tube. The dilution of the antibody will vary for each primary antibody used.
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14. Add 0.5 mL of the primary antibody solution to a clean well. Transfer the cell strainer containing the tissue into the primary antibody solution and place the plate on an orbital shaker for 48 h at 4°C.
Secondary Antibody Incubation
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15. Transfer the cell strainer containing the tissue to the next well in the plate that contains 0.5 mL of fresh wash solution. Place the plate on the orbital shaker for 15 min at room temperature.
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16. Repeat Step 15 for six total 15-min washes.
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17. Dilute secondary antibodies in antibody buffer in a 1.5-mL microcentrifuge tube on ice and mix by pipetting or inverting the tube. The dilution of the antibody will vary for each secondary antibody used.
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18. Add 0.5 mL of the secondary antibody solution to a clean well. Transfer the cell strainer containing the tissue into the secondary antibody solution, and place the plate on an orbital shaker for 48 h at 4°C.
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19. From this point onward, keep the plate covered to minimize exposure of the fluorophores to light.
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This can be achieved by covering the plate with the lid of a standard freezer box or by using aluminum foil.
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Tissue Mounting and Visualization
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20. Transfer the cell strainer containing the tissue to the next well in the plate that contains 0.5 mL of fresh wash solution. Place the plate on the orbital shaker to wash the tissue for 15 min at room temperature.
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21. Repeat Step 20 for six total 15-min washes.
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22. Add 0.5 mL of mounting medium to a clean well. Transfer the cell strainer containing the tissue into the well containing mounting medium and let it sit briefly.
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This step is to remove excess liquid that would dilute the mounting medium.
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23. Add 0.5 mL of mounting medium to another clean well. Transfer the cell strainer containing the tissue into the well containing mounting medium and let it sit for between 12 and 20 h at 4°C.
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24. Mount the tissue on slides in a small amount of fresh mounting medium. Elevate the coverslip by ∼150 µm to avoid distorting the tissue, which can be accomplished by creating a small pedestal with two additional coverslips. Transfer the tissue to the slide using a long Pasteur pipette and then position the tissue gently with forceps between the two pedestals. Gently lower the coverslip onto the pedestals.
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Be careful to suction the brain or VNC only into the first inch of the pipette, otherwise it may get stuck to the side and lost.
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25. Seal the slide with nail polish by sealing all of the exposed edges of the pedestal and the coverslip and allow it to dry. Be careful to avoid mixing the nail polish with mounting media or else it will not harden. Once the nail polish has completely hardened at room temperature, the slides can be placed in a slide holder and either stored at 4°C or you can proceed to Step 26. If using fluorescent secondary antibodies, it is recommended to image the tissue in the week after mounting, because the fluorescence will decay over time.
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26. View the whole-mount brain or VNC on a confocal microscope.
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For imaging the entire brain, we frequently use a 25×/0.8 numerical aperture immersion corrected objective, used with glycerol as the immersion medium to match the refractive index of the mounting medium most closely. We conduct a tiled scan with 10% overlap to capture the brain in four tiles that can be stitched together.
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We recommend imaging the brain using a microscope that is equipped with laser compensation that will allow the user to increase the laser intensity and photomultiplier tube gain as the imaging depth increases. Insect tissue in general, and mosquito tissue in particular, scatters light to a larger degree than mammalian tissue, so laser and gain compensation are particularly important when imaging whole-mount tissue.
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The boundaries of the brain and between regions can be used to generate three-dimensional reconstructions using both open-source and commercially available software. Ae. aegypti neuroanatomy resources that include brain reconstructions are available at mosquitobrains.org and insectbraindb.org (Matthews et al. 2019; Heinze et al. 2021).
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ACKNOWLEDGMENTS
We thank Olena Riabinina, Stanley Heinze, Nicolas Renier, and members of the Rockefeller University Bio-Imaging Resource Center and its staff members (RRID:SCR_017791) for help and advice. M.A.Y. is supported by funding from the Searle Scholars Program, the Richard and Susan Smith Family Foundation, the Esther A. & Joseph Klingenstein Fund, and the Simons Foundation.
Footnotes
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From the Mosquitoes collection, edited by Laura B. Duvall and Benjamin J. Matthews.
