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Preparation and Immunofluorescence Staining of the Trachea in Drosophila Larvae and Pupae
果蝇幼虫和蛹中气管的制备和免疫荧光染色   

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Abstract

The Drosophila melanogaster trachea is a branched network of rigid chitin-lined tubes that ramify throughout the body and functions as the fly’s respiratory organ. Small openings at the ends of the tracheal tubes allow gas exchange to occur by diffusion between internal tissues and the exterior environment. Tracheal tubes are lined by a single layer of epithelial cells, which secrete chitin and control tube morphology and size. Studies of tracheal development in Drosophila embryos have elucidated fundamental mechanisms of tube morphogenesis and maintenance in vivo, and identified major signaling pathways that regulate these processes (Manning and Krasnow, 1993; Affolter and Shilo, 2000; Zuo et al., 2013; Kerman et al., 2006; Schottenfeld et al., 2010). In recent years, there has been growing interest in the trachea during metamorphosis, when tracheal branches that had served as the respiratory organ in the larva decays and is repaired or replaced by new tracheal tissue arising from committed tracheal progenitor cells, or mature tracheal cells de-differentiated to a progenitor state (Manning and Krasnow, 1993; Sato and Kornberg, 2002; Guha et al., 2008; Guha, and Kornberg, 2005; Weaver and Krasnow, 2008; Pitsouli and Perrimon, 2010; Chen and Krasnow, 2014) forming the adult tracheal by the end of the process. The ongoing decay and tissue formation models aspects of tissue repair and regeneration in other organisms, and has been used to understand how progenitor cells divide and differentiate (Pitsouli and Perrimon, 2010; Pitsouli and Perrimon, 2013), and how they grow out of their niche to replace decaying tissue (Chen and Krasnow, 2014). Here, we present a protocol to dissect, fix, and immunostain tracheal tissue in Drosophila larvae and pupae undergoing metamorphosis. This protocol can be used to immunostain proteins expressed in tracheal tissue, or to amplify signals from weakly expressed fluorescent reporters (as shown in Figure 6). With the appropriate antibodies and genetic reporters, this protocol can be used to visualize decaying larval trachea and the progenitor cells that replace them in a time-course analysis, as well as determine expression of proteins in these cells that may play a role in tissue decay and replacement.

Materials and Reagents

  1. 60 mm x 15 mm petri dish [i.e., Falcon® Petri dish (Corning, catalog number: 351007 )]
  2. Flat-bottom 4-well dish [i.e., Nunclon® Δ Multidishes, 4 wells, flat bottom (Sigma-Aldrich, catalog number: D6789-1CS )]
  3. Gold SealTM Rite-OnTM Frosted microslides (VWR International, Erie Scientific, catalog number: 3050 )
  4. Micro cover glasses (coverslips), 22 x 22 mm Square No. 1 (VWR International, catalog number: 48366-067 )
  5. Black electrical tape (i.e., 3M Scotch Super 33+ Vinyl Electrical Tape 0.75 in x 450 in)
  6. Clear nail polish [i.e., crystal clear (Sally Hansen Hard as nails polish)]
  7. KimwipesTM (4.4 x 8.4 in.) (Thermo Fisher Scientific, catalog number: 06-666 )
  8. Austerlitz Insect Pins® ,12 mm length x 0.10 mm diameter (Minutiens in stainless steel, size 0.10 mm) (Entomoravia)
  9. 10 μl, 200 μl and 1,000 μl pipet tips (USA Scientific, TipOne, catalog number: 1111-3000 , 1111-0000 and 1111-2021 )
  10. Disposable glass Pasteur pipettes (Corning, catalog number: 7095D-5x ) with 1 ml rubber bulbs (Sigma-Aldrich, catalog number: Z111589 )
  11. Aluminum foil
  12. Parafilm M® All-Purpose laboratory film (2" x 250') (VWR International, Bemis Company, catalog number: PM992 )
  13. Drosophila melanogaster larvae and pupae of desired genotype raised at 25 °C
  14. Vials and bottles with closures [FisherbrandTM stock bottles (catalog number: AS117 ), FisherbrandTM cotton balls (catalog number: 22-456-880 ), FisherbrandTM Drosophila products, BuzzPlugsTM (catalog number: AS277 ), FisherbrandTM Drosophila vials (catalog number: AS514 )] with Drosophila food (see Cold Spring Harbor Protocols, 2014)
  15. Dow Corning SYLGARD® 184 Silicone Elastomer Kit [184 SIL ELAST KIT 0.5 KG (Ellsworth Adhesives)]
  16. 4% paraformaldehyde (PFA) diluted in PBS [i.e., 16% paraformaldehyde (VWR International, catalog number: 100503-916 ) diluted to 4% in PBS]
  17. Primary antibody to stain protein of interest [i.e., Chicken-anti-GFP (Abcam, catalog number: ab13970 ) to stain tracheal-expressed GFP in ppk4-Gal4, UAS-GFP larvae and pupae]
  18. Fluorescence conjugated secondary antibody to visualize and amplify primary antibody staining [i.e., Alexa488-conjugated Goat-anti-Chicken (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11039 ) to stain the above anti-GFP primary antibody]
  19. Normal serum from the same species as the secondary antibody [i.e., normal goat serum (Vector laboratories, catalog number: S-1000 )]
  20. Vectashield® mounting media (Vector Laboratories, catalog number: H-1000 )
  21. NaCl
  22. KCl
  23. Na2HPO4
  24. KH2PO4
  25. 1x Phosphate-buffered saline (PBS) (see Recipes)
  26. TritonTM X-100 (Sigma-Aldrich, catalog number: X100 ) diluted to 0.1% in PBS (see Recipes)
  27. Block solution (see Recipes)
  28. DAPI staining solution (see Recipes)
  29. Dissection dish (see Recipes)

Equipment

  1. Incubator to house Drosophila set to 25 °C
  2. Stereomicroscope with light source [i.e., Carl ZeissTM StemiTM 2000C with KL 300 LED Cold Light Source (120 V) (Thermo Fisher Scientific, catalog number: 12-070-284 )]
  3. Dumont #5 mirror finish forceps biology tips/straight/inox/11 cm (Fine Science Tools, catalog number: 11252-23 )
  4. Vannas spring scissors-straight/sharp/8 cm/3 mm cutting edge (Fine Science Tools, catalog number: 15000-00 )
  5. Benchtop shaker (i.e., Bellco Glass 7744-06115 Mini Orbital Shaker)
  6. Clay AdamsTM Nutator Mixer (BD, catalog number: 421105 )
  7. P2, P20, P100, and P1000 Pipetman® Pipettes (Gilson Scientific Ltd., catalog number: F144801 , F123600 , F123615 and F123602 )
  8. Small watercolor paintbrush, round, size 0
  9. Stainless steel spatula with a micro spoon end (Ted Pella Inc., catalog number: 13500 )

Procedure

  1. Dissection of larvae and pupae by ventral filleting
    1. Pick larvae and pupae of the appropriate age. Tracheal progenitors are activated during the late second-instar (L2) stage and remodel the trachea throughout the third-instar (L3) and pupal stages.
      1. The staging of Drosophila melanogaster larvae and pupae is described in (Ashburner, 2005).
      2. Young (L1 to early L3 stage) larvae [see Ashburner (2005) for more information on larval staging] will burrow into the food as they feed. To isolate these larvae, scoop out a portion of the food with the spoon end of the spatula and place it into a petri dish filled with PBS. Gently separate the food with the spatula until larvae can be seen floating in the PBS, and then transfer the larvae into a new PBS-filled petri dish with a small paintbrush or a glass Pasteur pipette.
      3. Late L3 stage larvae that are about to enter puparium formation will migrate onto the walls of the vial or bottle they are cultured in, and because of this behavior are commonly referred to as wandering L3 larvae. Wet a small paintbrush by dipping briefly in PBS and blot the excess PBS on a KimwipeTM. Gently pick up larvae from the wall of the vessel with the tip of the brush. Dip the tip of the brush with the larvae into a petri dish filled with PBS and move the brush back and forth to allow the larvae to be transferred to the PBS.  
      4. During the pupal stages, tracheal metamorphosis occurs rapidly, and so it is necessary to obtain pupae of precise age. To do this, locate animals that have just entered pupariation [0 h after puparium formation (APF)]; they should be white, attached to the side of the vessel, and immobile (Ashburner, 2005) (Figure 1A). Gently detach these pupae with a small wet paintbrush and transfer them into a new vial. Set the pupae onto the walls of the vial so that the ventral surface of the animal is attached to plastic (the way they normally attach to the vessel wall).  Incubate at 25 °C until the desired time APF has been reached, then gently detach and pick up the pupae with a small wet paintbrush and transfer to a dissection dish.  When transferring, be careful not to damage the pupae by putting too much pressure on them.
    2. Preparations for dissection.
      1. Using forceps pick up insect pins one at a time and insert them vertically tip down into the silicone elastomer layer of the dissection dish to be used. Insert just deep enough that the pins are held in place by the silicone elastomer. This way, pins can be easily retrieved with forceps and moved to where they are needed during dissection.
    3. Ventral-filleting larvae, and pupae younger than 12 h APF (before eversion of the head) (see Videos 1 and 2).

      Video 1. Dissecting larva

      Video 2. Dissecting pupa younger than 12 h APF

      1. Place Petri dishes containing larvae on ice for 5 to 10 min. The cold temperature will slow the larva’s movements and facilitate dissection. Do not leave larvae on ice for more than 45 min. Pupae are immobile and will not need to be exposed to cold temperature prior to dissection.
      2. Fill a dissection dish with cold PBS and move the larva or pupa into the dissection dish.
      3. With forceps, turn the animal ventral side up, and gently hold it in place using forceps. Locate the posterior (Figure 2A and 3A, white arrows) of the left and right tracheal dorsal trunks, a set of large air-filled tubes running along the anterior-posterior axis on the dorsal side of the animal, and insert a pin between the trunks at the posterior (Figure 2A and 3A, white arrowheads) into the silicone elastomer. Insert another pin (Figure 2A and 3A, black arrowheads) between the dorsal trunks at the anterior end of the animal (Figure 2A and 3A, black arrows).  When dissecting larvae, first stretch the animal by pulling on the anterior with forceps, and then insert the second pin just below the pigmented mouthparts.   
      4. Gently pull up on the ventral epidermis (and cuticle in pupae) with forceps, and carefully insert the bottom blade of the Vannas spring scissors where the posterior pin has been inserted, and slide the blade just under the ventral epidermis. If necessary, enlarge the opening made by the pin by pulling on the epidermis with forceps before inserting the scissor blade. Cut towards the anterior of the animal (Figure 2A and 3A, horizontal dashed lines). Pupae can also be cut from anterior to posterior, although larvae are more easily cut from posterior to anterior.  
      5. Grasp the edges of the cut epidermis (and cuticle in pupae) with forceps, pull gently to the side and secure with pins (Figure 2B-C and 3B-C; asterisks) so that the epidermis is one flat layer (Figure 2C and 3C). Make additional cuts to the epidermis and cuticle as necessary (Figure 3A vertical dashed lines) to allow the sample to be pinned flat. Be careful to not pin or damage tracheal branches during the process.  
      6. Remove internal organs by pulling on them with forceps. Cut any terminal tracheal branches attached to the tissue (Figure 2D, arrows) so that the tissue can be removed.
      7. The main tracheal branches [as described in Manning and Krasnow (1993)] should now be visible (Figure 2C, E and 3C); they are rigid air-filled tubes and attached to the epidermis. Clear remaining debris and tissue around tracheal branches by gently pipetting PBS onto the sample with a Pasteur pipette.
    4. Ventral-filleting pupae older than 12 hours APF (after head eversion) (see Video 3).

      Video 3. Dissecting pupa older than 12 h APF

      1. Pupae that have completed the larval-pupal molt and everted their heads (Figure 1C), which occurs around 12 h APF, need to be removed from the pupal case prior to dissection. To do this, move the pupa onto a dry dissection dish ventral side down. Work under a stereomicroscope, and hold a pair of Dumont #5 forceps in each hand. Carefully hold the pupa with forceps using the non-dominant hand and use forceps in the dominant hand to insert the tip of an insect pin through the posterior end of the pupal case just below where the pupa has detached from the pupal case (Figure 4A, white arrowhead). Continue inserting the pin into the silicone elastomer layer of the dish; the pupa should be securely pinned to the dish. Using Vannas spring scissors make a horizontal cut through both layers of the pupal case at the anterior end above the pupa’s head (Figure 4A, vertical dashed line). Remove the rest of the pupal operculum with forceps. Gently insert scissors just under the pupal case along the anterior-posterior axis (careful not to damage the pupa inside), and cut the pupal case from anterior to the posterior (Figure 4A, horizontal dashed line). Carefully peel away the pupal case and remove it or pin it down to the dish (Figure 4C, asterisks), making extra cuts in the pupal case as necessary to relieve any pressure exerted by the case on the pupa inside. Gently pick up the pupa from the pinned case with a small wet brush and transfer to a new dissection dish. An alternative method to remove the pupa from the pupal case is described in Wang and Yoder (2011).  
      2. With a wet brush (or very gently with forceps) orient the pupa ventral side up.  Gently holding the pupa in place with forceps, insert a pin through the head (Figure 4D, black arrowhead).
      3. With forceps, pull up on the ventral epidermis at the opening made by the pin and slide the bottom blade of the Vannas spring scissors just under the ventral epidermis. Cut all the way to the posterior of the animal (Figure 4D, black dashed line).
      4. Make a small cut in the dorsal epidermis from posterior to anterior just to the midpoint of the abdomen (Figure 4D, black dotted line). This cut allows the pupa to be pinned flat.  
      5. Grasp the cut edges of the ventral epidermis with forceps and pin down so that the epidermis is one flat layer (Figure 4E).
      6. Much of the internal tissues of the pupa will be histolyzed and can be removed by gently pipetting PBS onto the sample with a Pasteur pipette. Pipetting in the anterior to posterior direction results in least amount of damage to the tracheal branches. Any remaining tissue can be gently pulled out with forceps.
    5. Fix the dissected larvae and pupae.
      1. Remove the PBS containing detached internal tissues with a P1000 pipet.  Gently pipet 1 to 2 ml of 4% PFA along the sides of the dish, enough to cover all of the samples.  
      2. Place the dissection dish on an orbital shaker in the fume hood. Cover samples with a light protected box if samples express fluorescent proteins. Set the shaker on a medium-low setting (i.e., 3 or 4) and allow the PFA to fix the samples at room temperature for 30 min.
    6. Remove the 4% PFA and properly dispose as hazardous waste. Gently pipet 1 to 2 ml room temperature PBS, and shake for 5 minutes to wash out residual PFA. Repeat 2 more times.  
    7. With forceps and working under the stereomicroscope, carefully remove the pins holding the samples in place. Transfer the fixed samples into a 4-well dish for staining.  Up to 6 larvae and 8 pupae can be stained in each well.   
    8. Proceed with the staining procedure described below or store the samples immersed in PBS at 4 °C with the plate wrapped in Parafilm. Samples can be stored for up to a few weeks.

  2. Immunofluorescence staining larval and pupal trachea
    1. Permeabilize the fixed sample(s) by incubating in 650 μl PBS with 0.1% TritonTM X-100 at room temperature for 5 min. Pipet in liquids gently along the sides of each well to prevent damaging the samples. If samples express a fluorescent reporter, cover the dish with aluminum foil or a light protected box for this and subsequent steps.
    2. Remove the PBS with 0.1% TritonTM X-100, add 650 μl block solution into each well, and incubate at room temperature for 30 min on a Nutator mixer.
    3. Dilute primary antibody in block solution. Remove the block solution from the step above and apply 650 μl of the primary antibody solution per well. If amount of the antibody solution needs to be conserved, add just enough to immerse the samples, about 250 μl per well. Incubate overnight at 4 °C on a Nutator mixer.  
    4. Remove the antibody solution, add 650 μl PBS 0.1% Triton, and incubate on a Nutator mixer for 5 min at room temperature to wash away residual antibodies. Remove wash solution, and repeat 2 more times.   
    5. Wash the samples 3 more times in 650 μl PBS 0.1% Triton for 30 min each. These longer washing steps are necessary to remove any antibodies that might have been trapped in the tracheal tubes.
    6. Dilute fluorescence conjugated secondary antibody in PBS 0.1% Triton, and apply 250 to 650 μl to each well. Incubate on a Nutator mixer for 1 h at room temperature.  Cover the dish with aluminum foil or a light protective box for this and subsequent steps.
    7. Wash the samples in PBS 0.1% Triton 3 times for 5 min each.  
    8. Apply 650 μl DAPI staining solution each well and incubate for 10 to 20 min.
    9. Remove the DAPI solution, and wash the samples with PBS 0.1% Triton 3 times for 5 min each, and then 3 times for 30 min each.

  3. Mount samples on glass slides
    1. Cut two pieces of black electrical tape about 20 mm x 5 mm and affix to the glass slide. Leave a space slightly less than the width of the coverslip between the two pieces of tape (Figure 5A). The pieces of tape are used to create a small space between the slide and the coverslip (Figure 5B); if the samples are mounted without the tape, the coverslip may crush the sample and damage tracheal structures.
    2. Add two to three drops of Vectashield® mounting media onto the glass slide between the two pieces of tape. Place your samples in the Vectashield® and adjust the orientation of the samples so that the internal surface of the sample containing the tracheal branches is on top and the exterior surface of the epidermis is on the bottom. To facilitate imaging, orient the samples on the slide so that when observing the slide through the microscope the anterior is on the left and the posterior on the right. With forceps, remove any dust particles that might be lodged in the sample.  
    3. Cover samples with a coverslip and allow the Vectashield® to fill the space between the slide and coverslip, gently push down on the coverslip with forceps if necessary. Remove any extra Vectashield leaking from under the coverslip with a Kimwipe.
    4. Apply clear nail polish around the edges of the coverslip to seal in the samples and Vectashield®. Cover the slides with a light protective box, and allow the nail polish to dry at room temperature (approximately 10 min).  
    5. Proceed with microscopic analysis, or store the slides at 4 °C.

Representative data



Figure 1. Appearance of Drosophila melanogaster pupae. A. Pupae that have just entered puparium formation (0 h APF) are white, immobile, and attached to the sides of the culture vessel. B. As the pupa ages, the cuticle tans and turns from white to a yellow brown color. C. Pupae undergoing the larval-pupal molt detach from the pupal case. The molt is completed by head eversion (around 12 h APF), and a gap can be seen between the pupal case and the head of the pupa inside (white arrow). Scale bar, 500 µm


Figure 2. Dissecting Drosophila melanogaster larvae by ventral filleting. A. Larvae are first pinned ventral side up between the tracheal dorsal tracheal trunks at the posterior end (white arrows; position of pin indicated by the white arrowhead). A second pin is inserted just under the pharynx at the anterior end (black arrowhead) between the left and right anterior dorsal trunks (black arrows). The ventral epidermis is then cut along the anterior-posterior axis (black dashed line). B. The cut epidermis is pulled to the side and pinned down to the dissection dish (asterisk). C. Internal tissues are pulled out by forceps; thin terminal branches of the trachea attached to the tissues are cut (arrows) to allow the tissue to be removed. D. As internal tissues are removed, more pins (asterisks) are inserted to pin the epidermis flat. The major branches of the trachea are visible after removal of the internal tissues (enlarged in E). DB, dorsal branch; DT, dorsal trunk; TC; transverse connective; LT, lateral trunk. Tracheal progenitor cells (arrow) in the fourth (Tr4) and fifth (Tr5) tracheal metameres are often visible in wandering L3 larvae, appearing as a small patch of tissue located at the junction of the TC with the DT. Scale bars, 500 µm (A-C), 250 µm (D-E)


Figure 3. Dissecting young pupae (younger than 12 h APF). A. Young pupae that have not yet completed the larval-pupal molt and everted their heads (0 to 12 h APF) are dissected by pinning at the anterior (black arrowhead) and posterior (white arrowhead) at a position between the left and right dorsal trunks (arrows). Cuts are then made through the pupal case and ventral epidermis (black dashed lines). B and C. Additional pins (asterisks) are inserted to pin the sample flat. Internal organs and tissues are removed by pulling with forceps and small cuts made by spring scissors. C. After removal of internal tissue, tracheal branches are visible. DB, dorsal branch; DT, dorsal trunk; TC; transverse connective; LT, lateral trunk. Scale bars, 500 µm


Figure 4. Dissecting older pupae (older than 12 h APF). A. Pupae that have everted their heads need to be removed from the pupal case. First a pin is inserted through the posterior end of the pupal case in the region where the pupa has detached from the case (white arrowhead) to secure the pupa to the dissection dish. Cuts are then made through the pupal case just above the head (vertical black dashed line) and through the dorsal pupal case from anterior to posterior (horizontal black dashed line). B. A pupal after the cuts through pupal case have been made. C. The pupal case is then peeled away and held down with pins (asterisks) until the pupa can be picked up with a brush and transferred to a new dissection dish. D. The pupa is oriented ventral side up, and a pin is inserted into the head (black arrowhead). Cuts through the ventral (black dashed line) and dorsal (black dotted line) epidermis are made and the epidermis is pinned down until it is flat, as shown in (E). Additional pins used to pin the sample down are indicated by asterisks. Internal tissues are removed by forceps or gentle pipetting of PBS onto the sample to expose the trachea. F. Main branches of the pupal trachea. DB, dorsal branch; DT, dorsal trunk; TC, transverse connective; LT, lateral trunk; PAT, pupal abdominal trachea. Scale bars, 500 µm (A-E), 250 µm (F)


Figure 5. Mounting samples on glass slides. A. To prevent the coverslip from crushing the tracheal in larval and pupal samples, two small pieces of electrical tape (arrows) are affixed to the slide. B. The tape props the coverslip up, creating a small space between the slide and coverslip.


Figure 6. Representative images of immunostained larvae and pupae. A. Tracheal metamere 5 (Tr5) of a third-instar ppk4-Gal4, UAS-GFP; btl-RFP-moe larva in which larval tracheal cells have been immunostained for the GFP reporter expressed in these cells (green), and outgrowing progenitor cells (arrowhead) are immunostained for the membrane-associated RFP reporter they express (red). Nuclei are stained with DAPI (blue). Dashed line outlines the progenitor niche; solid line indicates the niche exit. B. Tr5 and Tr6 in a btl-RFP-moe/ crumbs::GFP pupa 6 h after puparium formation. Signals from the RFP reporter expressed in progenitor cells and GFP expressed on the apical surface of trachea are amplified by immunostaining for RFP (red) and GFP (green), respectively. Expression of the Pruned SRF protein in trachea progenitors (arrowheads) is detected by an anti-Pruned antibody (white). Scale bar, 50 µm (A), and 100 µm (B). Image taken from a previously published study (Chen and Krasnow, 2014).

Notes

  1. Young larvae (first and young second instar larvae) may be too small to be dissected by the method described in this protocol.
  2. Tracheal tubes can sometimes trap antibodies, leading to false positive staining. Try to include negative controls in experiments, such as samples incubated with secondary antibody but not primary antibody, or samples that do not express the epitope detected by the primary antibody.
  3. Steps B3 to B5 can be skipped if using a primary antibody directly conjugated to a fluorophore.
  4. Staining of the tracheal lumen with a fluorescence-labeled chitin binding probe or wheat germ agglutinin (Weaver and Krasnow, 2008; Chen and Krasnow, 2014) can be accomplished with this protocol, skipping steps B3 to B5.
  5. The lumens of tracheal branches autofluoresce when observed through the UV channel. This autofluorescence can be used to determine outlines of the tracheal branches, and is distinguishable from nuclear DAPI stains. However, to avoid confusing antibody stains with the autofluorescence, secondary antibodies conjugated to fluorescent reporters that fluoresce at other wavelengths should be used.
  6. Alternative detergent solutions (i.e., 0.1% Tween-20 diluted in PBS) can be used to permeabilize cell membranes in the samples. Permeabilizing samples with 0.1% Triton X-100 was chosen because it was optimal for staining both the tracheal lumen and tracheal cells. 0.1% Tween-20 resulted in good staining in tracheal cells but weak staining in tracheal lumens.

Recipes

  1. Recipe for 1x phosphate-buffered saline (PBS)
    NaCl            8 g
    KCl              0.2 g
    Na2HPO4    1.44 g  
    KH2PO4      0.24 g
    Dissolve in 800 ml H2O, adjust pH to 7.4, add H2O to 1 L final volume.
  2. 0.1% TritonTM X-100 PBS solution
    Extract 5 ml TritonTM X-100 with a 6 ml syringe and mix into 45 ml of PBS
    Vortex until completely dissolved to make 10% TritonTM X-100 stock solution
    Dilute 2.5 ml 10% stock solution in PBS (250 ml final volume) to make 0.1% solution
  3. Block solution
    0.1% TritonTM X-100 PBS solution
    10% normal serum from the species of the secondary antibody (i.e., normal goat serum)
  4. DAPI staining solution
    0.1 µg/ml DAPI diluted in PBS
  5. Dissection dish
    Mix components of the SYLGARD® 184 Silicone Elastomer Kit following the manufacturer's instructions
    Apply enough of the mixture to the bottom of a 60 mm x 15 mm Petri dish to cover the bottom of the dish with a 1 cm thick layer
    Let the mixture cure overnight at room temperature before use

Acknowledgments

This protocol is adapted from methods developed by others and used in previous studies (Weaver and Krasnow, 2008; Pitsouli and Perrimon, 2010; Pitsouli and Perrimon, 2013; Levi et al., 2006). Funding was provided by the Howard Hughes Medical Institute (HHMI), a Genentech Graduate Fellowship, and a Ruth L. Kirschstein NIH training grant.

References

  1. Affolter, M. and Shilo, B. Z. (2000). Genetic control of branching morphogenesis during Drosophila tracheal development. Curr Opin Cell Biol 12(6): 731-735.
  2. Ashburner, M., Golic, K. G.and Hawley, R. S. (2005). Chapter 6: Life Cycle. In: Ashburner, M. (ed). Drosophila: a laboratory handbook. Cold Spring Harbor Laboratory Press, pp 150-158.
  3. Chen, F. and Krasnow, M. A. (2014). Progenitor outgrowth from the niche in Drosophila trachea is guided by FGF from decaying branches. Science 343(6167): 186-189.
  4. Cold Spring Harbor Protocols (2014). Fly Food
  5. Guha, A. and Kornberg, T. B. (2005). Tracheal branch repopulation precedes induction of the Drosophila dorsal air sac primordium. Dev Biol 287(1): 192-200.
  6. Guha, A., Lin, L. and Kornberg, T. B. (2008). Organ renewal and cell divisions by differentiated cells in Drosophila. Proc Natl Acad Sci U S A 105(31): 10832-10836.
  7. Kerman, B. E., Cheshire, A. M. and Andrew, D. J. (2006). From fate to function: the Drosophila trachea and salivary gland as models for tubulogenesis. Differentiation 74(7): 326-348.
  8. Levi, B. P., Ghabrial, A. S. and Krasnow, M. A. (2006). Drosophila talin and integrin genes are required for maintenance of tracheal terminal branches and luminal organization. Development 133(12): 2383-2393.
  9. Manning, G. and Krasnow, M. A. (1993). In: Martinez-Arias, A. and Bate, M. (eds). The Development of Drosophila. Cold Spring Harbor Laboratory Press, 609-685.
  10. Sato, M. and Kornberg, T. B. (2002). FGF is an essential mitogen and chemoattractant for the air sacs of the Drosophila tracheal system. Developmental cell 3(2): 195-207.
  11. Schottenfeld, J., Song, Y. and Ghabrial, A. S. (2010). Tube continued: morphogenesis of the Drosophila tracheal system. Curr Opin Cell Biol 22(5): 633-639.
  12. Pitsouli, C. and Perrimon, N. (2010). Embryonic multipotent progenitors remodel the Drosophila airways during metamorphosis. Development 137(21): 3615-3624.
  13. Pitsouli, C. and Perrimon, N. (2013). The homeobox transcription factor cut coordinates patterning and growth during Drosophila airway remodeling. Sci Signal 6(263): ra12.
  14. Wang, W. and Yoder, J. H. (2011). Drosophila pupal abdomen immunohistochemistry. J Vis Exp 56: e3139.
  15. Weaver, M. and Krasnow, M. A. (2008). Dual origin of tissue-specific progenitor cells in Drosophila tracheal remodeling. Science 321(5895): 1496-1499.
  16. Zuo, L., Iordanou, E., Chandran, R. R. and Jiang, L. (2013). Novel mechanisms of tube-size regulation revealed by the Drosophila trachea. Cell Tissue Res 354(2): 343-354.

简介

melanogaster 气管是一种由几丁质内衬的管组成的分支网络,分布在整个身体并作为苍蝇的呼吸器官。气管管端部的小开口允许通过内部组织和外部环境之间的扩散而发生气体交换。气管管由单层上皮细胞衬里,其分泌几丁质和对照管形态和尺寸。在Drosop hila 胚胎中的气管发育的研究阐明了管形态发生和维持体内的基本机制,并且鉴定了调节这些过程的主要信号传导途径(Manning和Krasnow,1993; Affolter和Shilo,2000; Zuo等人,2013; Kerman等人,2006; Schottenfeld等人, em,2010)。近年来,在变态过程中对气管的兴趣日益增长,当在幼虫中用作呼吸器官的气管分支被修复或被由致动的气管祖细胞产生的新的气管组织或成熟的气管细胞(Manning和Krasnow,1993; Sato和Kornberg,2002; Guha等人,2008; Guha和Kornberg,2005; Weaver和Krasnow,2008; Pitsouli和Perrimon, 2010; Chen和Krasnow,2014)在过程结束时形成成人气管。正在进行的衰变和组织形成模拟其他生物体中组织修复和再生的方面,并且已经用于理解祖细胞如何分裂和分化(Pitsouli和Perrimon,2010; Pitsouli和Perrimon,2013),以及它们如何从它们的利基来替代腐烂组织(Chen和Krasnow,2014)。在这里,我们提出解剖,修复和免疫染色的果蝇幼虫和蛹变态的气管组织的协议。该协议可用于免疫染色在气管组织中表达的蛋白质,或用于扩增来自弱表达的荧光报道分子的信号(如图6所示)。使用适当的抗体和遗传报告物,该方案可用于显示衰变的幼虫气管和在时间过程分析中替代它们的祖细胞,以及确定这些细胞中可能在组织衰变中起作用的蛋白质的表达和更换。

材料和试剂

  1. 60mm×15mm培养皿[即 Falcon 培养皿(Corning,目录号:351007)]
  2. 平底4孔培养皿[即NunclonMultidishes,4孔,平底(Sigma-Aldrich,目录号:D6789-1CS)]
  3. Gold Seal?Rite-On?磨砂微滑块(VWR International,Erie Scientific,目录号:3050)
  4. 微盖玻片(盖玻片),22×22mm方形1(VWR International,目录号:48366-067)
  5. 黑色电子胶带(即 3M Scotch Super 33+乙烯基电子胶带0.75 in x 450 in)
  6. 清除指甲油[即清澈透明(Sally Hansen Hard as nails polish)]
  7. Kimwipes TM(4.4×8.4英寸)(Thermo Fisher Scientific,目录号:06-666)
  8. Austerlitz Insect Pins ,12mm长×0.10mm直径(Minutiens in stainless steel,size 0.10mm)(Entomoravia)
  9. 10μl,200μl和1000μl移液管吸头(USA Scientific,TipOne,目录号:1111-3000,1111-0000和1111-2021)
  10. 一次性玻璃巴斯德吸管(Corning,目录号:7095D-5x),带有1ml橡皮球(Sigma-Aldrich,目录号:Z111589)
  11. 铝箔
  12. 全部用途的实验室薄膜(2"×250')(VWR International,Bemis公司,目录号:PM992)
  13. 黑腹果蝇在25℃下培养的所需基因型的幼虫和蛹
  14. 装有瓶盖的小瓶和瓶[Fisherbrand TM原液瓶(目录号:AS117),Fisherbrand TM棉球(目录号:22-456-880),Fisherbrand TM果蝇产品,BuzzPlugs TM(目录号:AS277),Fisherbrand TM (参见Cold Spring Harbor Protocols,2014)的果蝇(Drosophila)小瓶(目录号:AS514)
  15. 道康宁SYLGARD ? 184硅氧烷弹性体套件[184 SIL ELAST KIT 0.5 KG(Ellsworth Adhesives)]
  16. 在PBS中稀释的4%多聚甲醛(PFA)[即16%多聚甲醛(VWR International,目录号:100503-916),在PBS中稀释至4%]
  17. 用于染色感兴趣的蛋白质的初级抗体[例如鸡抗GFP(Abcam,目录号:ab13970),用于在ppk4-Gal4,UAS-GFP 中染色气管表达的GFP,幼虫和蛹]
  18. 荧光共轭二级抗体以显现和扩增一级抗体染色[即Alexa488缀合的山羊抗鸡(Thermo Fisher Scientific,Invitrogen ,目录号:A-11039)染色上述抗GFP初级抗体]
  19. 来自与第二抗体相同物种的正常血清(即正常山羊血清(Vector laboratories,目录号:S-1000)]
  20. Vectashield 安装介质(Vector Laboratories,目录号:H-1000)
  21. NaCl
  22. KCl
  23. Na HPO 4
  24. KH 2 PO 4
  25. 1x磷酸盐缓冲盐水(PBS)(参见配方)
  26. 在PBS中稀释至0.1%的Triton X-100(Sigma-Aldrich,目录号:X100)(见Recipes)
  27. 块解决方案(参见配方)
  28. DAPI染色溶液(见配方)
  29. 解剖菜(见配方)

设备

  1. 用于容纳果蝇的孵育器设置为25℃
  2. 具有光源的立体显微镜[即,具有KL 300 LED冷光源(120V)(Thermo Fisher Scientific,目录号:12-070-284)的Carl Zeiss TM Stemi TM 2000C]
  3. Dumont#5镜面镊子生物学提示/直/inox/11厘米(Fine Science Tools,目录号:11252-23)
  4. Vannas弹簧剪刀直线/锐利/8厘米/3毫米切削刃(Fine Science Tools,目录号:15000-00)
  5. 台式振动台(即 Bellco Glass 7744-06115迷你轨道振动台)
  6. Clay Adams TM Nutator Mixer(BD,目录号:421105)
  7. P2,P20,P100和P1000 Pipetman移液管(Gilson Scientific Ltd.,目录号:F144801,F123600,F123615和F123602)
  8. 小水彩画笔,圆形,尺寸0
  9. 具有微匙端的不锈钢刮铲(Ted Pella Inc.,目录号:13500)

程序

  1. 通过腹侧切片解剖幼虫和蛹
    1. 选择适当年龄的幼虫和蛹。气管祖细胞在晚期第二阶段(L2)阶段期间被激活,并在整个第三阶段(L3)和蛹阶段重塑气管。
      1. 在(Ashburner,2005)中描述了黑腹果蝇幼虫和蛹的分期。
      2. 幼虫(L1至早期L3期)幼虫[参见Ashburner(2005)有关幼虫分期的更多信息]将在它们进食时钻进食物中。为了隔离这些幼虫,用铲子的勺子端捞出一部分食物,并将其放入装有PBS的培养皿中。轻轻地用刮刀分开食物,直到幼虫可以漂浮在PBS中,然后将幼虫转移到一个新的PBS填充的陪替氏培养皿,用小画笔或玻璃巴斯德吸管。
      3. 即将进入蛹形成的晚期L3期幼虫将迁移到它们在其中培养的小瓶或瓶的壁上,并且因为这种行为通常被称为徘徊L3幼虫。通过在PBS中短暂浸泡来润湿小画笔,并在Kimwipe TM上印迹多余的PBS。用刷子的尖端轻轻地从容器的壁上拿起幼虫。将刷子的尖端与幼虫一起放入装有PBS的培养皿中,并且来回移动刷子,以允许幼虫转移到PBS。  
      4. 在蛹期,气管变态迅速发生,因此有必要获得精确年龄的蛹。为此,定位刚刚进入瞳孔的动物[蛹形成后0小时(APF)];它们应该是白色的,附着在容器的侧面,并且是不动的(Ashburner,2005)(图1A)。轻轻地用一个小的湿画笔分离这些蛹,并将它们转移到一个新的小瓶。将蛹设置在小瓶的壁上,使动物的腹侧表面附着??塑料(它们通常连接到血管壁的方式)。在25°C孵育,直到达到所需的时间APF,然后轻轻地分离,并用小的湿画笔拿起蛹,并转移到解剖菜。转移时,小心不要对蛹造成太大的压力,以免损坏蛹。
    2. 解剖准备。
      1. 使用镊子拿起昆虫针一次一个,并将它们垂直插入到要使用的解剖碟的有机硅弹性体层。插入恰好足够深,以使销通过硅氧烷弹性体保持在适当位置。这样,销可以容易地用镊子取回,并移动到需要的地方,在解剖。
    3. 腹圆片幼虫和蛹小于12小时APF(头外翻之前)(见视频1和2)。

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      视频1.剖析幼虫
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      视频2.解剖年龄小于12小时的蛹APF
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      1. 在冰上放置含有幼虫的培养皿5至10分钟。冷的温度会减缓幼虫的运动,便于分离。不要将幼虫放在冰上超过45分钟。蛹是不动的,并且不需要在解剖之前暴露于冷的温度。
      2. 用冷PBS填充解剖盘,将幼虫或蛹移入解剖盘。
      3. 用镊子,将动物腹侧朝上,轻轻地用钳子保持在位。找到左和右气管背侧干的后部(图2A和3A,白色箭头),一组大型充气管沿着动物背侧前后轴运行,并插入一个销在(图2A和3A,白色箭头)进入硅氧烷弹性体。在动物前端的背侧躯干之间插入另一个针(图2A和3A,黑色箭头)(图2A和3A,黑色箭头)。当解剖幼虫时,首先用镊子拉动前面的动物,然后将第二个针插入色素口腔下方。  
      4. 用镊子轻轻地拉起腹侧表皮(和蛹中的角质层),并且小心地插入已插入后针的万那斯弹簧剪刀的底部刀片,并将刀片滑动到腹侧表皮下。如果需要,在插入剪刀片之前用镊子拉动表皮来扩大针的开口。朝动物的前面切割(图2A和3A,水平虚线)。蛹也可以从前向后切割,尽管幼虫更容易从后向前切割。  
      5. 用钳子抓住切割的表皮(和蛹中的角质层)的边缘,轻轻地拉到侧面并用针固定(图2B-C和3B-C;星号),使得表皮是一个平坦层(图2C和3C )。根据需要对表皮和角质层进行额外切割(图3A的垂直虚线),以允许样品固定平坦。小心不要在过程中固定或损坏气管分支。  
      6. 用镊子拉动内脏器官。切除附着到组织的任何末端气管分支(图2D,箭头),使得组织可以被去除。
      7. 主要的气管分支(如Manning和Krasnow(1993)所述)现在应该是可见的(图2C,E和3C);它们是刚性的空气填充管并附接到表皮。通过用巴斯德吸管轻轻吸取PBS到样品上,清除气管分支周围的残留碎屑和组织。
    4. 腹部剔除蛹超过12小时APF(头外翻后)(见视频3)。

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      视频3.剖析大于12小时APF的蛹
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      1. 已经完成幼虫蛹蜕皮和外翻的蛹(图1C),其发生在约12小时APF,需要在解剖之前从蛹的情况中去除。为此,将蛹移到干燥的夹层皿腹侧。在立体显微镜下工作,并在每只手上握着一对Dumont#5镊子。使用非优势手用镊子小心地握住蛹,并且使用优势手中的镊子将昆虫针的尖端插入通过蛹的后端,刚好在蛹已经从蛹壳分离的地方(图4A,白色箭头)。继续将销插入盘的硅氧烷弹性体层;蛹应该牢固地固定在盘子上。使用Vannas弹簧剪刀在蛹头部上方的前端(图4A,垂直虚线)处穿过蛹壳的两层进行水平切割。用镊子除去蛹的其余部分。轻轻地沿着前后轴将镊子下方的小剪刀(小心不要损坏蛹内)剪刀,并从前面切割蛹蛹到后面(图4A,水平虚线)。小心地剥离蛹胴体并将其移除或固定到盘上(图4C,星号),根据需要在蛹的情况下额外切割以减轻由壳体施加在蛹上的任何压力。轻轻拿起蛹从固定的情况下用一个小的湿刷,并转移到一个新的解剖菜。在Wang和Yoder(2011)中描述了从蛹的病例中去除蛹的替代方法。  
      2. 用湿刷子(或非常轻轻地用镊子)使蛹的腹侧朝上。轻轻地用镊子夹住蛹,在头部插入一个针(图4D,黑色箭头)。
      3. 用镊子,拉起在针的开口的腹侧表皮,滑动Vannas春天剪刀的底部刀片刚好在腹侧表皮下。一直切到动物的后面(图4D,黑色虚线)。
      4. 在背部表皮从后到前只是到腹部的中点(图4D,黑色虚线)做一个小切口。这种切割允许蛹被钉扎平。  
      5. 用镊子和针向下抓住腹侧表皮的切缘,使表皮是一个平层(图4E)。
      6. 蛹的大部分内部组织将被组织溶解并且可以通过用巴斯德吸管轻轻地将PBS移液到样品上来除去。从前到后方向的移液导致对气管分支的最小量的损伤。任何剩余的组织可以用镊子轻轻拉出
    5. 固定解剖的幼虫和蛹。
      1. 用P1000移液管取出含有分离的内部组织的PBS。轻轻地吸取1至2毫升的4%的PFA沿盘的侧面,足以覆盖所有的样品。  
      2. 将解剖盘放在通风橱中的轨道摇床上。如果样品表达荧光蛋白,用光保护盒盖上样品。将振动器设置为中低档( 3或4),并让PFA在室温下固定样品30分钟。
    6. 取出4%PFA,并妥善处置为危险废物。轻轻吸取1到2毫升室温PBS,摇动5分钟,洗出残余的PFA。重复2次以上。  
    7. 用钳子和在立体显微镜下工作,小心地移除固定样品的销。将固定样品转移到4孔培养皿中染色。每个孔最多可以染6条幼虫和8条蛹。  
    8. 继续下面描述的染色程序或将样品浸入4℃的PBS中,将样品包在石蜡膜中。样品可储存长达几周。

  2. 免疫荧光染色幼虫和蛹气管
    1. 通过在室温下在含有0.1%Triton TM X-100的650μlPBS中孵育5分钟来使固定的样品透过。沿着每个孔的边轻轻地吸取液体,以防止损坏样品。如果样品表达荧光报道分子,用铝箔或光保护盒盖上这个盘子,然后进行后续步骤
    2. 用0.1%Triton TM sup-X-100除去PBS,向每个孔中加入650μl封闭溶液,并在Nutator搅拌器上在室温下孵育30分钟。
    3. 稀释一抗在封闭溶液中。从上述步骤中移除封闭溶液,每孔加入650μl一抗溶液。如果抗体溶液的量需要保守,加入刚好足以浸渍样品,每孔约250μl。在4℃下在Nutator混合器上孵育过夜。  
    4. 取出抗体溶液,加入650μlPBS 0.1%triton,并在Nutator混合器上室温孵育5分钟以洗去残留的抗体。取出洗涤溶液,并重复2次以上。  
    5. 在650μlPBS 0.1%triton中洗涤样品3次,每次30分钟。这些更长的洗涤步骤对于除去可能已经被捕获在气管管中的任何抗体是必需的。
    6. 在PBS 0.1%triton中稀释荧光共轭二抗,并对每个孔应用250至650μl。在室温下在Nutator混合器上孵育1小时。用铝箔或光保护盒盖住菜肴,以便进行此步骤和后续步骤
    7. 在PBS 0.1%triton中洗涤样品3次,每次5分钟。  
    8. 每孔加入650μlDAPI染色溶液,孵育10?20分钟
    9. 取出DAPI溶液,用PBS 0.1%triton洗涤样品3次,每次5分钟,然后洗涤3次,每次30分钟。

  3. 在玻璃载玻片上安装样品
    1. 切割两片黑色电磁带约20毫米x 5毫米,并贴在载玻片上。留出稍小于两片胶带之间的盖玻片的宽度的空间(图5A)。胶带片用于在载玻片和盖玻片之间产生小空间(图5B);如果样品没有安装胶带,盖玻片可能会破碎样品并损坏气管结构。
    2. 将两到三滴Vectashield ?安装介质添加到两片胶带之间的载玻片上。将样品放在Vectashield ?中,并调整样品的方向,使含有气管分支的样品的内表面在顶部,表皮的外表面在底部。为了便于成像,将样品定向在载玻片上,使得当通过显微镜观察载玻片时,前面在左边,后面在右边。使用镊子,清除可能存在于样品中的任何灰尘颗粒。  
    3. 用盖玻片覆盖样品,并允许Vectashield ?填充载玻片和盖玻片之间的空间,如果必要,用镊子轻轻地向下推盖玻片。删除任何额外的Vectashield泄漏从盖玻片下与Kimwipe。
    4. 在盖玻片边缘涂上清晰的指甲油,以密封样品和Vectashield 。用光保护盒盖住幻灯片,并允许指甲油在室温下干燥(约10分钟)。  
    5. 继续进行显微镜分析,或将载玻片存储在4°C

代表数据



图1. 黑腹果蝇蛹的外观 A.刚刚进入蛹形成(0小时APF)的蛹是白色的,不动的,附着在培养物的两侧船只。 B.随着蛹的老化,角质层变白并从白色变成黄棕色。 C.幼虫经历幼虫蛹蜕皮从蛹的情况。通过头部外翻(大约12小时APF)完成蜕皮,并且可以在蛹的情况和蛹内部的头部之间看到间隙(白色箭头)。比例尺,500μm


图2.通过腹侧切片解剖黑腹果蝇幼虫。 A.幼虫首先被固定在腹侧,在后端的气管背侧干(白色箭头;针的位置由白色箭头指示)。第二个销插入在左前和右前背侧躯干之间的前端(黑色箭头)的咽下正下方(黑色箭头)。然后沿前后轴切割腹侧表皮(黑色虚线)。 B.切割的表皮被拉到侧面并固定到解剖碟(星号)。 C.用镊子取出内部组织;切割附接到组织的气管的薄末端分支(箭头)以允许组织被移除。 D.当去除内部组织时,插入更多的销(星号)以将表皮销平。在移除内部组织后(在E中放大),气管的主要分支是可见的。 DB,背支; DT,背脊; TC;横向连接; LT,外侧躯干。在第四(Tr4)和第五(Tr5)气管metameres中的气管祖细胞(箭头)在徘徊的L3幼虫中经常可见,表现为位于TC与DT的接合处的小组织的组织。比例尺,500μm(A-C),250μm(D-E)


图3.剖析幼蛹(小于12小时APF)。A.通过钉扎分离尚未完成幼虫 - 蛹蜕皮和翻转的幼蛹(0至12小时APF)的幼蛹(黑色箭头)和后部(白色箭头)在左右背部躯干(箭头)之间的位置。然后通过蛹壳和腹侧表皮进行切口(黑色虚线)。 B和C。插入附加的销(星号)以固定样品平面。通过用镊子和由弹簧剪刀制成的小切口拉动来移除内部器官和组织。 C.去除内部组织后,气管分支是可见的。 DB,背支; DT,背脊; TC;横向连接; LT,外侧躯干。比例尺,500μm


图4.剖析较早的蛹(12岁以上的APF)。A.已经翻转过的蛹需要从蛹中移除。首先,在蛹从盒(白色箭头)分离的区域中将销插入通过蛹壳的后端,以将蛹固定到解剖碟。然后通过刚好在头部上方的蛹的情况(垂直黑色虚线)和通过背部瞳孔情况从前面到后面(水平黑色虚线)进行切割。 B.蛹后通过蛹的情况下。 C.然后将蛹胴体剥离并用针(星号)压住直到蛹被刷子拿起并转移到新的解剖皿中。 D.蛹朝向腹侧向上,针插入头(黑色箭头)。切割通过腹部(黑色虚线)和背部(黑色虚线)表皮,并且表皮被钉扎直到它是平的,如(E)所示。用于固定样品的附加引脚用星号表示。通过镊子或PBS轻轻移液到样品上以移除内部组织以暴露气管。 F.蛹气管的主要分支。 DB,背支; DT,背脊; TC,横向连接; LT,外侧干; PAT,瞳孔腹气管。比例尺,500μm(A-E),250μm(F)


图5.将样品安装在载玻片上。为了防止盖玻片破碎幼虫和蛹样品中的气管,将两个小的电子胶带(箭头)固定到载玻片上。 B.胶带支持盖玻片,在幻灯片和盖玻片之间创建一个小空间。


图6.免疫染色的幼虫和蛹的代表性图像。A.第三龄期ppk4-Gal4,UAS-GFP的气管周围5(Tr5) btl-RFP-moe幼虫,其中幼虫气管细胞已经对在这些细胞中表达的GFP报道分子进行免疫染色(绿色),并且长出的祖细胞(箭头)针对其表达的膜相关RFP报告分子进行免疫染色(红色)。用DAPI(蓝色)染色细胞核。虚线概述了祖先的利基;实线表示利基出口。 B.Trr和Tr6在蛹形成6小时后的btl-RFP-moe/crumbs :: GFP蛹中。来自在祖细胞中表达的RFP报道分子的信号和在气管顶端表面上表达的GFP分别通过RFP(红色)和GFP(绿色)的免疫染色扩增。通过抗剪切抗体(白色)检测修剪的SRF蛋白在气管祖细胞(箭头)中的表达。比例尺,50μm(A)和100μm(B)。图片取自以前发表的研究(Chen和Krasnow,2014)。

笔记

  1. 幼虫(第一和幼年第二龄幼虫)可能太小,不能通过本协议中所述的方法解剖。
  2. 气管管有时可以捕获抗体,导致假阳性染色。尝试在实验中包括阴性对照,例如用二抗但不是一抗孵育的样品,或不表达由一抗检测的表位的样品。
  3. 如果使用直接缀合到荧光团的第一抗体,可以跳过步骤B3至B5。
  4. 用荧光标记的几丁质结合探针或小麦胚芽凝集素染色气管腔(Weaver和Krasnow,2008; Chen和Krasnow,2014)可以用该方案完成,跳过步骤B3至B5。
  5. 当通过UV通道观察时,气管分支的腔自发荧光。这种自发荧光可用于确定气管分支的轮廓,并且可与核DAPI染色区分开。然而,为了避免将抗体染色与自体荧光混淆,应当使用与在其他波长处荧光的荧光报告物偶联的二抗。
  6. 可以使用替代的洗涤剂溶液(即在PBS中稀释的0.1%Tween-20)来渗透样品中的细胞膜。选择具有0.1%Triton X-100的透性样品,因为其对于染色气管腔和气管细胞是最佳的。 0.1%Tween-20在气管细胞中导致良好的染色,但在气管腔中染色较弱

食谱

  1. 1×磷酸盐缓冲盐水(PBS)的配方
    NaCl            8克
    KCl              0.2 g
    Na HPO 4 1.44克
    KH 2 PO 4       0.24克
    溶解在800ml H 2 O中,将pH调节至7.4,加入H 2 O至1L最终体积。
  2. 0.1%Triton X-100PBS溶液
    用6ml注射器提取5ml Triton TM X-100,并混合入45ml PBS中
    涡旋直到完全溶解,以制备10%Triton TM sup-X-100储备溶液 稀释2.5ml 10%储备溶液在PBS(250ml终体积)中以制备0.1%溶液
  3. 封锁解决方案
    0.1%Triton X-100PBS溶液
    来自第二抗体(即正常山羊血清)的10%正常血清
  4. DAPI染色溶液
    0.1μg/ml用PBS稀释的DAPI
  5. 解剖刀
    按照制造商的说明混合SYLGARD ? 184硅氧烷弹性体组件的组分
    将足够的混合物施加到60mm×15mm培养皿的底部,以覆盖具有1cm厚的层的培养皿的底部。
    让混合物在使用前在室温下固化过夜

致谢

该协议改编自其他人开发的并在先前研究中使用的方法(Weaver和Krasnow,2008; Pitsouli和Perrimon,2010; Pitsouli和Perrimon,2013; Levi等人,2006)。资金由霍华德休斯医学研究所(HHMI)提供,基因技术研究生奖学金和Ruth L. Kirschstein NIH培训补助金。

参考文献

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  2. Ashburner,M.,Golic,K.G.and Hawley,R.S。(2005)。第6章:生命周期。在:Ashburner,M。(ed)。 果蝇:实验室手册。 Cold Spring Harbor Laboratory Press,pp 150-158
  3. Chen,F。和Krasnow,M.A。(2014)。 祖细胞在果蝇气管中的生长因子由FGF指导衰变 343(6167):186-189。
  4. 冷泉港协议(2014)。 Fly Food
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  7. Kerman,B.E.,Cheshire,A.M。和Andrew,D.J。(2006)。 从命运到功能:果蝇气管和唾液腺作为管腔形成的模型 < em>区分 74(7):326-348。
  8. Levi,B.P.,Ghabrial,A.S.and Krasnow,M.A。(2006)。 果蝇 talin和整合素基因是维持气管末端分支所必需的,发展 133(12):2383-2393。
  9. Manning,G.and Krasnow,M.A。(1993)。 In:Martinez-Arias,A.and Bate,M。(eds)。 <果蝇的发育。 Cold Spring Harbor Laboratory Press,609-685
  10. Sato,M。和Kornberg,T.B。(2002)。 FGF是果蝇的气囊的必需的促分裂原和趋化因子气管系统。 发育细胞 3(2):195-207
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  12. Pitsouli,C。和Perrimon,N。(2010)。 胚胎多潜能祖细胞在变态过程中重塑了果蝇航空公司。 137(21):3615-3624
  13. Pitsouli,C。和Perrimon,N。(2013)。 同源异型框转录因子在果蝇气道重塑过程中切割坐标图案和生长。 Sci Signal 6(263):ra12。
  14. Wang,W。和Yoder,J.H。(2011)。 果蝇蛹的腹部免疫组织化学。 em> J Vis Exp 56:e3139。
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引用:Chen, F. (2016). Preparation and Immunofluorescence Staining of the Trachea in Drosophila Larvae and Pupae. Bio-protocol 6(9): e1797. DOI: 10.21769/BioProtoc.1797.
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