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מאמר שיטה

Immunostaining to Visualize Viral Antigen and Host Protein Colocalization in the Insect Gut

127 צפיות

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31 ביולי 2026

במאמר זה

תקציר

Source: Zhang, L., et al. Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts. J. Vis. Exp. (2021)

The video demonstrates the preparation of gut tissue from a plant virus–infected insect vector for fluorescence imaging. Fixed guts were washed, permeabilized, and incubated with two fluorescent antibodies in a blocking solution—one targeting a viral antigen in vesicles, the other a vesicle-associated membrane protein. The tissue was counterstained for actin filaments, mounted with a nuclear stain, and imaged to reveal in situ colocalization of viral and host proteins.

פרוטוקול

1. Nonviruliferous insect rearing

  1. Collect white-backed planthoppers (WBPHs) from rice fields and rear with rice seedlings in 1 L glass beakers covered with insect-proof net in an incubator at 28 °C with 16 h light and 8 h dark. Because southern rice black-streaked dwarf virus (SRBSDV) is not transmitted via eggs, newly hatched nymphs are not viruliferous.
  2. With a brush pen, gently brush insects from the beaker, rearing insects into a new beaker of fresh rice seedlings each week until WBPH nymphs have hatched. Continue rearing these hatched nonviruliferous nymphs to 2- or 3-instar.
    NOTE: Brush carefully to avoid WBPHs flying from the beaker or damaging them.

2. Virus acquisition and collection of viruliferous insects

  1. Transfer nonviruliferous insects from the glass beakers onto fresh SRBSDV-infected rice plants covered with an insect-proof net for a 2-day virus-acquisition access period (AAP) by feeding on plants. Then, collect the insects in glass beakers containing fresh rice seedlings.
  2. After 2 days, collect the insects from the glass beakers with a manual aspirator for dissection and excision of the gut.
    NOTE: The minimum AAP of SRBSDV is 5 min for both WBPH nymphs and adults, but the insects should be allowed to feed on fresh SRBSDV-infected rice plants for 2 days to achieve the acquisition efficiency of up to 80%.

3. Reagent preparation

  1. Dissolve 8.5 g of sodium chloride (NaCl), 3.5 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), and 0.25 g of sodium dihydrogen phosphate (NaH2PO4) in 1 L of double-distilled water (ddH2O) to prepare a 0.01 M solution of phosphate-buffered saline (PBS).
  2. Add 4 g of paraformaldehyde to 100 mL of PBS to prepare 4% (m/v) paraformaldehyde in PBS.
  3. Add 2 mL of Triton X-100 into 98 mL of PBS to prepare 2% (v/v) Triton X-100.

4. Dissection of adults and excision of guts

  1. Use a pipettor to place 100 µL of PBS on a glass slide. Place the slide on the stage of an optical microscope.
  2. Collect the SRBSDV-infected adults from glass beakers with a manual aspirator and place them in 1.5 mL tubes. Place the tubes on ice to paralyze insects, and then transfer a paralyzed adult into the 100 µL of PBS on the slide with the abdomen up.
    NOTE: The insects will be thoroughly paralyzed after 5 min on ice.
  3. Use tweezers in one hand to clamp the body, and then remove the head with another set of tweezers in the other hand.
  4. Clamp the sides of the abdomen with one set of tweezers and clamp the ovipositor or the copulatory organ of the tail with the other set. Then, pull away the intersegmental membrane of one abdominal segment carefully and slowly expose the gut in the abdomen.
  5. Continue tearing away the membrane and gradually pull out the complete gut from the abdomen. Gently pull off the tail, which is connected to the end of the gut, to remove the complete gut.
    NOTE: Pull very carefully or the gut will be damaged.
  6. Place excised guts into a 200 µL centrifuge tube, add 200 µL of PBS to the tube, and gently suck-release the solution with a pipette to wash the guts thoroughly.

5. Dissection of nymphs and excision of guts

NOTE: Nymph bodies are more fragile than adult bodies, and the gut is easily damaged when pulled from the tail. Therefore, the most reliable method to excise the nymph gut is by pulling from the head.

  1. Use a pipettor to place 100 µL of PBS on a glass slide. Place the slide on the stage of an optical microscope.
  2. Collect the SRBSDV-infected nymphs from glass beakers with a manual aspirator and place them into 1.5 mL tubes. Place the tubes on ice to paralyze insects. Then, transfer a paralyzed nymph into the 100 µL of buffer on the slide with the abdomen facing up.
  3. Use tweezers to detach the tail of the nymph. Then clamp the insect body to fix gently and use the other set to clamp the head. Gently pull the head away from the body while still maintaining its attachment to the gut so that the head is detached from the body, but the gut is still attached to the thorax and abdomen.
    NOTE: After the head is detached, the corpus adiposum of the nymph will flow out, making the PBS turbid. Remove the turbid PBS solution and change with 100 µL of fresh PBS.
  4. With the tweezers still clamping the body, use the other set to move the head carefully, and gradually pull out the gut.
  5. Gently detach the gut from the head with tweezers, and eventually obtain an intact gut without damaging the body of the planthopper.
  6. Place excised guts into a 200 µL centrifuge tube, add 200 µL of PBS to the tube, and gently suck-release the solution with a pipette to wash the guts thoroughly.
    NOTE: The excised guts should be cleaned well with PBS to remove any contaminating fat bodies from the abdominal cavity; they can interfere with the staining protocol.

6. Labeling protocols for SRBSDV virions and an insect protein

  1. Prepare the antibodies and mounting medium used in this assay. Antibodies are anti-SRBSDV antibody labeled with Dylight 549 (red) against SRBSDV virions, anti-VAMP7 antibody labeled with Dylight 488 (green) against the insect protein, vesicle-associated membrane protein 7 (VAMP7), Dylight 633 phalloidin (blue), and mounting medium containing 4,6-diamidino-2-phenylindole (DAPI, blue).
  2. Place the freshly excised and PBS-washed WBPH guts immediately in 100 µL of 4% (m/v) paraformaldehyde in a 200 µL centrifuge tube and hold for 2 h at room temperature.
    NOTE: The freshly excised WBPH guts should not be soaked in PBS for a longer duration, or the epithelial cells will be damaged.
  3. Remove 4% (m/v) paraformaldehyde with a pipettor, and then add 200 µL of PBS into the 200 µL centrifuge tube. After 10 min, remove PBS using a pipettor to eliminate any paraformaldehyde.
  4. Repeat this PBS wash step twice.
  5. Remove the PBS and add 200 µL of nonionic detergent Triton X-100 (2%, v/v). Permeabilize the samples in the nonionic detergent for 30 min at room temperature.
  6. Remove 2% (v/v) Triton X-100 with a pipettor, and then wash away any remaining detergent with three 10-minute washes with 200 µL of PBS (see steps 6.3 and 6.4).
  7. Dilute anti-SRBSDV antibody labeled by Dylight 549 (red) and anti-VAMP7 antibody labeled by Dylight 488 (green) 1:50 with 50 µL of bull serum albumin (3%, m/v).
  8. Add the diluted antibodies to the tube and incubate the samples overnight at 4 °C.
  9. Remove the antibody diluent with a pipettor, and then wash away the remaining antibody diluent with three 10 min washes with 200 µL of PBS.
  10. Dilute 1 µL of Dylight 633 phalloidin with 50 µL of PBS.
  11. Add 50 µL of diluted phalloidin to the tube and incubate the samples for 2 h at room temperature.
  12. Remove the phalloidin diluent with a pipettor, and then wash away the remaining phalloidin with three 10-minute washes with 200 µL of PBS.
    NOTE: Thorough washes are critical to reduce the background and nonspecific binding.
  13. Place a drop of mounting medium containing DAPI on a microscope slide and transfer the guts to the medium.
    NOTE: Gently unfold each gut with tweezers and avoid creating bubbles. There are about 15 guts per slide.
  14. Gently place a cover glass over the samples without creating bubbles.
    NOTE: The slide should be held at 4 °C in the dark to inhibit fluorescence quenching before observation with a laser scanning confocal microscope.
  15. View all samples with a laser scanning confocal microscope. Capture the images using blue light and save the files on a computer.

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חומרים

רשימת החומרים שנעשה בהם שימוש במאמר זה
שםחברהמספר קטלוגהערות
3% Bull serum albumin (BSA)CoolaberSL1331Dilute antibodies
Cover glassSolarbioYA0771-18*18mmFor slide making
Dissecting microscopeBeitjaXTL-7045B1For insect dissection
Laser scanning confocal microscopeZeissZeiss LSM880Observe fluorescence signal
Microscope slidesSolarbioZBP-7105For slide making
Mounting medium with 4'6-diamidino-2-phenylindole (DAPI)AbcamAB104139Label cell necleus
ParaformaldehydeSigma158127For tissues fixation
PhalloidinInvitrogenA22284Label actin of midgut epithiels
Triton X-100Amresco0290C484For tissues permeation
Tweezers (5-SA)AsOne6-7905-40For insect dissection

תגיות

סימון אימונופלואורסצנטיזיהוי אנטיגן נגיפירקמת מעי של חרקמיקרוסקופיה קונפוקליתוקטור של נגיף צמחיםחלבון ממברון של שלפוחיתצביעת סיבי אקטיןאינקובציית נוגדניםפרמיאביליזציה של רקמה
Immunostaining to Visualize Viral Antigen and Host Protein Colocalization in the Insect Gut
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