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zPACT: Tissue Clearing and Immunohistochemistry on Juvenile Zebrafish Brain
zPACT: 幼年斑马鱼脑的组织清除和免疫组化检测   

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Abstract

In studies of brain function, it is essential to understand the underlying neuro-architecture. Very young zebrafish larvae are widely used for neuroarchitecture studies, due to their size and natural transparency. However, this model system has several limitations, due to the immaturity, high rates of development and limited behavioral repertoire of the animals used.

We describe here a modified version of the passive clearing technique (PACT) (Chung et al., 2013; Tomer et al., 2014; Yang et al., 2014; Treweek et al., 2015), which facilitates neuroanatomical studies on large specimens of aquatic species. This method was initially developed for zebrafish (Danio rerio) (Frétaud et al., 2017; Mayrhofer et al., 2017; Xavier et al., 2017), but has also been successfully tested on other fish, such as medaka (Oryzias latipes) (Dambroise et al., 2017), Mexican cave fish (Astyanax mexicaus) and African zebra mbuna (Metriaclima zebra), and on other aquatic species, such as Xenopus spp. (Xenopus laevis, Xenopus tropicalis) (Fini et al., 2017). This protocol, based on the CLARITY method developed and modified by Deisseroth’s laboratory and others (Chung et al., 2013; Tomer et al., 2014; Yang et al., 2014), was adapted for use in aquatic species, including zebrafish in particular (zPACT).

This protocol is designed to render zebrafish specimens optically transparent while preserving the overall architecture of the tissue, through crosslinking in a polyacrylamide/formaldehyde mesh. Most of the lipids present in the specimen are then removed by SDS treatment, to homogenize the refractive index of the specimen by eliminating light scattering at the water/lipid interface, which causes opacity. The final clearing step, consists of the incubation of the specimen in a fructose-based mounting medium (derived from SeeDB) (Ke et al., 2013), with a refractive index matching that of the objective lens of the microscope. The combination of this technique with the use of genetically modified zebrafish in which green fluorescent protein (GFP) is expressed in specific cell populations provides opportunities to describe anatomical details not visible with other techniques.

Keywords: PACT(PACT), Tissue clearing(组织清除), Deep imaging(深度成像), Zebrafish(斑马鱼), Immunohistochemistry(免疫组化检查), Confocal microscopy(共聚焦显微镜)

Background

Adult teleost fish (zebrafish, medaka) are increasingly being used as model vertebrates in studies extending from stem cells to brain development (Than-Trong and Bally-Cuif, 2015; Dambroise et al., 2017), in all aspects of physiological and medical research (Oksanen et al., 2013) and in studies of complex types of behavior, such as learning, memory and addiction (Bailey et al., 2015).

In its role as a scientific service provider, the Tefor Core Facility has developed a reliable tissue clearing technique for dense, neuronal tissues of several millimeters in diameter, to satisfy the demand for studies of large specimens of aquatic animals.

By creating a set of neuroanatomical atlases of different developmental stages, this protocol will serve as a resource for other laboratories wishing to integrate their data into these anatomical reference frameworks (ARFs). At the time of publication, the prototype of a 7dpf ARF is available from https://www.zebrafish.tefor.net/. The data accessible via this URL will subsequently be consolidated into an annotated 3D neuroanatomy atlas and enriched with standardized high-resolution confocal imaging data for different developmental stages. The Tefor Core Facility is currently developing ARFs for 5 dpf, 6 dpf, 7 dpf larval heads and juvenile brains. The development of ARFs for other stages will depend on the demand from members of the zebrafish research community.

The integration of new specimens into the corresponding ARF requires precise staging of the developmental stage of the specimen and the use of DiI stain, as described here. This dye is used as a fiducial marker for the computational alignment of 3D imaging data into the corresponding ARF. However, the protocol can also be run without these steps if a direct comparison of the generated data with the existing data pool is not required.

As a compromise between specimen size and maturity, we focused on juvenile zebrafish brains during the development of this protocol. Juvenile zebrafish brains are smaller than those from older fish, but may be considered mature (Filippi, 2010). Moreover, the use of sexually immature animals should reduce data variability due to sexual dimorphism (Ampatzis, 2012). The smaller size of these animals also makes the protocol faster, because all its steps are dependent on the penetration of the applied agents into the specimens. However, the animals used have nevertheless reached a sufficiently advanced developmental stage for extrapolation of the findings to older specimens.

Materials and Reagents

  1. Sterile 50 ml conical tubes (Corning, catalog number: 430291 )
  2. Sharp blade (single-sided razor blade (e.g., Razor Blade Company, catalog number: 62-0167 ) or appropriate scalpel blade (e.g., #10))
  3. 94 x 16 mm Petri dishes (Greiner Bio One International, catalog number: 632102 )
  4. Small spatula for manipulating anesthetized fish during staging
  5. Sterile 15 ml conical tubes (Corning, catalog number: 431470 )
  6. Paintbrush (e.g., Kolinsky France, Manet 630, size 3 )
  7. 60 x 15 mm Petri dishes (Greiner Bio One International, catalog number: 628160 )
  8. Multipolymer protective gloves (Shield Scientific, catalog number: 66 9253 )
  9. Parafilm M (Sigma-Aldrich, Parafilm, catalog number: P7543 )
  10. Glass tubes for a rotisserie hybridization oven (Fisher Scientific, catalog number: 11791436 )
  11. 2 ml Eppendorf tube (STARLAB INTERNATIONAL, catalog number: E1420-2000 )
  12. 3.2 ml transfer pipette (Carl Roth, catalog number: EA67.1 )
  13. 2 ml glass tubes (Carl Roth, catalog number: H303.1 )
  14. 0.22 µm-pore filter (SARSTEDT, catalog number: 83.1826.001 )
  15. 6-week-old zebrafish raised under standard conditions (density, feeding, etc.; Lawrence and Mason, 2012; Lawrence et al., 2016)
  16. Rotifers (Brachionus plicatilis)
  17. Brine shrimps (Artemia nauplii)
  18. Dry food (Skretting, Gemma Micro)
  19. Pure nitrogen (Air Liquide, ALPHAGAZ 1)
  20. Alexa Fluor 488-conjugated secondary antibody, goat anti-chicken IgY (H+L) (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11039 )
  21. DiIC18(3) (Thermo Fisher Scientific, InvitrogenTM, catalog number: D282 )
  22. Dimethyl sulphoxide (DMSO) (Carl Roth, catalog number: 4720.1 )
  23. Primary antibody directed against GFP (chicken) (Aves Lab, catalog number: GFP-1020 )
  24. Low-melting point agarose (Sigma-Aldrich, catalog number: A4018 )
  25. Mineral oil (Carl Roth, catalog number: HP50.3 )
  26. Tricaine mesylate (MS222) (Sigma-Aldrich, catalog number: A5040 )
  27. Sodium bicarbonate (Carl Roth, catalog number: 0965.2 )
  28. 20x PBS (Santa Cruz Biotechnology, catalog number: sc-362183 )
  29. 16% formaldehyde (w/v), methanol-free (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28908 )
  30. 4% formaldehyde solution, methanol-free (w/v) (PFA) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28908 )
  31. Tween 20 (Sigma-Aldrich, catalog number: P7949 )
  32. 40% acrylamide solution in H2O (Sigma-Aldrich, catalog number: 01697 )
  33. VA-044 initiator (Wako Pure Chemical Industries, catalog number: 017-19362 )
  34. SDS pellet (Carl Roth, catalog number: CN30.3 )
  35. Boric acid (Carl Roth, catalog number: 6943.1 )
  36. 20x SSC (Carl Roth, catalog number: 1054.1 )
  37. Formamide (CH3NO) (Carl Roth, catalog number: 6749.3 )
  38. H2O2 solution (30%) (Carl Roth, catalog number: CP26.5 )
  39. Sodium azide (Sigma-Aldrich, catalog number: S2002 )
  40. Normal goat serum (NGS) (Antibodies, catalog number: 54-410 )
  41. Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
  42. D(-)-fructose (Carl Roth, catalog number: 4981.6 )
  43. SYLGARD 184 (Sigma-Aldrich, catalog number: 761036 )
  44. 10x MS222 solution (see Recipe 1)
  45. Fixing solution (4% PFA, 0.01 M PBS, 0.1% Tween 20) (see Recipe 2)
  46. Sylgard-coated Petri dish (see Recipe 3)
  47. PBST (1x PBS, 0.01% Tween 20) (see Recipe 4)
  48. Hydrogel solution (see Recipe 5)
  49. SDS solution (8% SDS, 0.2 M boric acid, pH 8.5) (see Recipe 6)
  50. Depigmentation (see Recipe 7)
    1. SSC 0.5x-Tween 20
    2. Depigmentation solution
  51. Storage solution (see Recipe 8)
  52. Blocking solution (see Recipe 9)
  53. Staining solution (see Recipe 10)
  54. Fructose-based high-refractive index solution (fbHRI) (see Recipe 11)

Equipment

  1. 3 fish tanks/line (e.g., Tecniplast 1 L Breeding Tank without the internal part)
  2. Laminated measuring grid (see Figure 1)
  3. Fume hood
  4. Ice bath & refrigerator
  5. Embryo spoon (Carl Roth, catalog number: TL85.1 )
  6. Dumont tweezers, straight (World Precision Instruments, catalog number: 500336 )
  7. Dumont tweezers, straight (World Precision Instruments, catalog number: 14099 )
  8. Dumont tweezers, 45° angle (World Precision Instruments, catalog number: 500234 )
  9. Stereomicroscope (Olympus, model: SZX10 )
  10. Fiber light source (Olympus, model: KL 2500 LED )
  11. Heated vacuum desiccator (VWR, SELECTA, catalog number: SELE4000474 )
  12. Hotplate (Labexchange, Bioblock Scientific, model: AM3001K )
  13. Vacuum pump (Savant, model: Gel Pump GP 110 )
  14. Histology processing cassettes (Simport, model: M503-12 )
  15. 400 ml beaker (VWR, catalog number: 213-1125 )
  16. 600 ml beaker (VWR, catalog number: 213-1126 )
  17. Rotisserie hybridization oven (Grant Boekel, model: HIR 1011 )
  18. 3D rocker Polymax 1040 (Heidolph Instruments, catalog number: 0 36130320 )
  19. Refractometer (KRUSS Optronic, catalog number: DR 301-95 )
  20. Leica TCS SP8 laser scanning microscope (Leica Microsystems, model: Leica TCS SP8 )
    1. 2 photomultipliers for the simultaneous detection of two channels
    2. Leica HC FLUOTAR L 25x/1.00 IMM motCorr objective (Leica Microsystems, catalog number: 15507703 )
  21. Heavy magnetic stirring bar (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F37118-0002 )

Software

  1. Fiji/ImageJ (http://fiji.sc)
  2. Amira (https://www.fei.com/software/amira-3d-for-life-sciences/)
  3. ITK-snap (http://www.itksnap.org)

Procedure

  1. Rearing and staging
    Fish are reared for 6 weeks under controlled conditions (photoperiod = 14 h/10 h light/dark; temperature = 26.5 ± 1 °C; pH = 7.8 ± 0.1; conductivity = 240 ± 30 µS/cm, NH4+ = 0 mg/L, NO2- = 0 mg/L, NO3- < 50 mg/L), in groups of 50 individuals per 1.4 L tank. They are fed on rotifers (Brachionus plicatilis; > 1,000/fish/day; one dose at noon) for the first two weeks of feeding, and then with brine shrimps (Artemia nauplii; ~250/fish/day) and dry food (Skretting, Gemma Micro; to apparent satiation, two doses, morning, afternoon) (Lawrence and Mason, 2012; Lawrence et al., 2016). All procedures are performed in accordance with European Union Directive 2010/63/EU.
    As suggested in a previous study (Parichy et al., 2009), the stage of interest (juvenile) is defined on the basis of the disappearance of the fin-fold, at about 5-6 weeks in local standard conditions, corresponding to a body length of 12.5 mm.
    The measuring and staging steps are performed under potentially lethal anesthesia and outside of water. These steps must therefore be performed rapidly, individually and very carefully, to ensure that all the fish survive the procedure. Fish of the desired size should be killed directly before fixation, to prevent a decay of expression patterns in the specimens. Measuring/staging should be performed in the fish facility, so these animals must be kept alive until they are transferred to the lab and killed and fixed under a fume hood. Fish that are too small can be left for a few days and then remeasured, and fish that are too large can be introduced into other experiments or the breeding program.
    1. Prepare the anesthetic solution: 200 ml of 1x MS222 solution, by diluting 10x MS222 solution (see Recipe 1) 1:9 in fish water in a fish tank (anesthesia tank).
    2. For each fish line, prepare two additional fish tanks:
      1. Name of the fish line + correct size.
      2. Name of the fish line + incorrect size.
    3. Anesthetize one fish at a time by transferring it to the anesthesia tank.
      Wait until the fish is completely anesthetized; test this by touching the tail with a spatula and checking for movement.
    4. Position the fish directly on the measuring grid (Figure 1).
      1. Remove the liquid surrounding the fish to avoid lens effects.
      2. With the spatula, position the mouth of the fish against the gray middle line and the back along the horizontal line, as shown in Figure 1.


        Figure 1. Staging juvenile fish by age and measuring their body length: Diagram of a fish on the measuring grid. The different color lines correspond to different distances from the center lines of the measuring grid: inner gray line 11 mm; magenta line 12 mm; cyan line 12.5 mm; yellow line 13 mm; outer gray line 14 mm. The fish in this figure would be considered to have a body length of 12.5 mm (central gray line to the cyan line). Zebrafish image modified from Togo picture gallery maintained by Database Center for Life Science (DBCLS).

    5. Measure the distance from the mouth to the point at which the tail begins.
      Note: Juvenile zebrafish typically measure 12.5 mm.
    6. Place the fish in the tank corresponding to its size.
      1. Fish that are too small can be harvested later.
      2. Fish that are too large can be used for later stages or breeding/stock maintenance.
    7. Check if the fish recovers fully from anesthesia.
      After 3-5 min, the fish should start swimming normally again. Swimming may be sluggish at first, but the fish should recover fully within 5-10 min.

  2. Zebrafish brain dissection
    1. Preparation of specimens and solutions
      1. Under the fume hood, prepare the fixing solution (4% PFA, 1x PBS, 0.1% Tween 20, see Recipe 2). Prepare 40 ml of fixing solution in a 50 ml conical tube for the prefixation of 5 fish heads. Keep the tubes at 4 °C.
      2. Kill the specimens in accordance with local regulations (e.g., European Union Directive 2010/63/EU, as developed in the recommendations of the Karlsruhe Institute of Technology for ‘The Humane Killing of Zebrafish’).
    2. Prefixation of heads
      1. Transfer specimens to 1x PBS and cut the head behind the gills (keep the operculum intact), with a sharp blade to avoid any compression or tearing of the CNS.
      2. Place the head in a 50 ml conical tube containing fixing solution (transfer with tweezers).
      3. Incubate at 4 °C for 18 h.
    3. Dissection
      1. Under the fume hood, prepare fresh fixing solution. Prepare 40 ml of fixing solution in a 50 ml conical tube for 5 fish brains. Keep the tubes at 4 °C.
      2. Use tweezers to transfer a prefixed head to an SYLGARD-coated Petri dish (see Recipe 3) filled with PBST (see Recipe 4).
      3. Under a stereomicroscope, carefully extract the brain from head with fine tweezers. This step requires fine motor skills and it may take some training and practice to develop a satisfactory dissection technique. (see Supplemental Video)
      4. Remove the surrounding tissues from the brain with ultra-fine tweezers.
        Notes:
        1. Avoid touching the brain with the tweezers by using the connective tissues as handles.
        2. Avoid tearing the brain by applying only tangential forces while peeling away connective tissues.
      5. Use an embryo spoon to transfer the dissected brain to the 50 ml conical tube containing fixing solution.
      6. Incubate at 4 °C for 5 h.
      7. Proceed to the next step of hydrogel incubation or keep the brain in storage solution.
    Note: For a detailed description of the dissection process we provide a dissection movie (see Supplemental Video).

  3. Tissue clearing
    1. Incubation in hydrogel solution
      1. Under the fume hood, prepare the hydrogel solution on ice (see Recipe 5).
        Note: The hydrogel solution must be prepared fresh, immediately before sample transfer into the solution.
      2. Keep the samples on ice throughout the experiment.
      3. Transfer the samples into a 15 ml Falcon tube with a fine paintbrush or a cut transfer pipette.
        Maximum: 8 brains/tube
      4. Remove as much liquid as possible from the 15 ml tube.
      5. Add 14 ml of hydrogel solution to the 15 ml tube containing the samples.
        Incubate at 4 °C for 48 h. 
    2. Polymerization
      1. Under the fume hood, set up the desiccator and set the hotplate to 37 °C.
      2. Label a 60 mm Petri dish with the name and number of the sample to be treated.
      3. Place the Petri dish on the 37 °C hotplate.
      4. Pour all 14 ml of hydrogel solution containing the brains into the Petri dish.
      5. Keep the Petri dish open and uncovered.
      6. Close the desiccator, ensuring a hermetic fit.
      7. Remove air with the vacuum pump until the pressure gauge reads -50 cm Hg.
      8. Carefully refill with pure nitrogen until the pressure gauge reads 0.
      9. Repeat the last two steps 6 times.
      10. Allow the samples to polymerize for 2.5 h.
    3. Passive clearing in SDS solution
      1. Follow these steps after polymerization for brains:
        1. Place clean cassettes in a 60 mm Petri dish containing 8% SDS solution (see Recipe 6).
        2. Wearing multipolymer protective gloves, use curved tweezers to remove each brain individually from the polymerized hydrogel and place it in your hand.
        3. Using a fine, soft paintbrush and 8% SDS solution, gently clean the brain, removing all of the surrounding hydrogel.
        4. Place the brains in the cassettes.
        5. Close the cassettes and place them in a 300 ml beaker containing 8% SDS solution.
      2. Cover the beaker with Parafilm and protect it from light for 2-5 h at room temperature, to dialyze out the remaining PFA, initiator and monomer.
      3. Place the cassettes in autoclaved rotisserie hybridization oven glass tubes containing 8% SDS solution.
      4. Incubate the hybridization tubes at 37 °C, with rotation, in the hybridization oven.
      5. Check the transparency of the samples every day (Figure 2).
        Note: The standard time for clearing is 8 days for juvenile brains.
      6. Once the sample has been successfully cleared (Figure 2), wash it three times in PBST. Replace the washing solution once daily for two more days.
      7. If the samples are not destined for use immediately after the third day of washing, store them in storage solution (see Recipe 8) at 4 °C.


        Figure 2. Time-series of the tissue clearing process: passive clearing of juvenile zebrafish brains (as described in steps C3c-C3e) is achieved within 8 days. This figure illustrates the clearing progress of juvenile fish brains in 8% SDS over time, showing an increase in transparency from: A. day 0, B. day 2, C. day 6, D. day 8. Scale bar = 1 mm.

  4. Depigmentation
    1. Pour 10 ml of pre-incubation solution (SSC 0.5x-Tween 20, see Recipe 7) in a 60 mm Petri dish and transfer the samples to the Petri dish with a paintbrush or embryo spoon.
      Note: Make sure you limit the dilution of the pre-incubation solution with PBST.
    2. Incubate at room temperature for 1 h.
    3. Under the fume hood, prepare the depigmentation solution (see Recipe 7) immediately before use.
    4. Again under the fume hood, remove the samples with some liquid (to ensure that they don’t dry out) and place them in the lid of the Petri dish. Remove all of the pre-incubation solution from the dish and replace it with depigmentation solution. Return the samples to the dish with a minimum of pre-incubation solution.
      Note: Keep the Petri dish uncovered and under light to accelerate the depigmentation process.
    5. Check the progress of the depigmentation every 10 min. Cover the Petri dish when you check the depigmentation progress under a stereomicroscope.
      Note: The standard depigmentation time is 45 min for juvenile brains.
    6. Once the depigmentation is complete, rinse twice sequentially in PBST under a fume hood.
      Note: Leave the rinsed samples on the bench overnight or for a minimum of 4 h before proceeding with the next step.

  5. Post-fixation
    1. Place the samples in a 2 ml Eppendorf tube. Remove all the liquid and add 1 ml of fixing solution.
    2. Incubate overnight at 4 °C.
      Notes:
      1. If the samples are to be used immediately after this step, rinse them four times in PBST for 3 h.
      2. If the samples are not going to be used immediately, remove the fixing solution and replace it with 1 ml of storage solution. Store the samples at 4 °C until use.

  6. Whole-mount labeling
    1. Transfer the samples to 2 ml glass tubes with a cut transfer pipette. Maximum: 5 brains/tube.
    2. To prevent dilution of the next solution, remove as much liquid as possible from the tube.
    3. Add 1 ml of blocking solution (see Recipe 9) to each tube.
    4. Incubate overnight at 4 °C.
    5. Remove the blocking solution and add 600 µl of staining solution (see Recipe 10) per tube.
    6. Add 1.2 µl of anti-GFP antibody per tube, to obtain a 1/500 dilution.
      Note: Other primary antibodies have been successfully tested. But the dilution must be optimized, primary antibody concentration generally ranges from 1 µg/ml to 10 µg/ml.
    7. Incubate at room temperature for 7 days, with gentle agitation, on a 3D rocker.
    8. Rinse 3 times, for 20 min each, in PBST.
    9. Remove all the liquid from the tube and add 600 µl of staining solution per tube.
    10. Add 1.5 µl of Alexa Fluor 488-conjugated secondary antibody per tube, to obtain a 1/400 dilution.
    11. Incubate at room temperature for 5 days, with gentle shaking, on a 3D rocker.
    12. Rinse for 2 days in several changes of PBST.
      1. Optional: DiI staining is used as a fiducial marker. Incubate the samples for 2 days in 600 µl of staining solution per tube at 1 µM dilution.
        Note: Prepare 1 mM Dil stock solution by dissolving DiI powder in DMSO.
      2. Rinse the DiI-stained sample in PBST for a minimum of 3 h.

  7. Mounting and imaging
    1. Preparation for imaging
      1. Incubate the samples in 50% fbHRI/50% diluent for at least 6 h (see Recipe 11 and Figure 3B)
      2. Incubate in fbHRI for at least 12 h (Figure 3C).
    2. For imaging, mount samples in a droplet of 1% (wt/vol) low-melting point agarose in a 60 mm Petri dish, and cover them generously (to a depth of about 10 mm) with fbHRI.
    3. To avoid evaporation during prolonged imaging, cover with a layer of mineral oil.
    4. Whole-mount brain fluorescence can be imaged with a Leica TCS SP8 laser scanning confocal microscope equipped with a Leica HC FLUOTAR L 25x/1.00 IMM motCorr objective. The fluorescence signal is detected by exciting the fluorophores with lasers at wavelength of 488 and 552 nm, at a laser power of 1-3%, with capture by two internal photomultipliers (PMT). Tile-scan imaging followed by mosaic stitching is required to capture images of the whole brain.


      Figure 3. Final clearing of juvenile zebrafish brains after passive clearing by homogenizing the refractive index in fructose based high refractive index medium (fbHRI). A. After replacing the SDS solution of the passive clearing process with PBS the tissue turns opaque again. B. After incubation in 50% fbHRI in PBS the opacity is significantly reduced. C. After incubation in 100% fbHRI, the tissue clearing process is finished. Scale bar = 1 mm.

Data analysis

The analysis of imaging data depends strongly on the scientific questions addressed. Many methods are therefore available. The tissue-clearing techniques described here were used by Dambroise et al. (2017) to visualize maximum intensity projections of parts of complete specimens in Fiji. Manual and machine-assisted segmentations of expression patterns, and visualization of the resulting areas can be achieved in ITK-snap or Amira (FEI). Typical large specimens treated with the tissue clarification technique described here can be analyzed as shown in Video 1, a translation movie, in which each slice of the original stack is converted into a movie frame. Standard mp4-compression reduced the file size by several orders of magnitude, facilitating access.


Video 1. Translation movie of a juvenile zebrafish brain after tissue clearing, anti-GFP and DiI staining along the optical axis: each slice of the confocal stack was converted into a frame of this movie. Translation direction from ventral to dorsal; caudal to the right. The bounding box dimensions of the underlying stack are 3.2 x 2 x 1.5 mm, the isotropic voxel size is 1.74 µm. Original bit depth, 12 bits. Green: ETvmat2: GFP expression pattern; Magenta: DiI fiducial staining.

Recipes

  1. 10x MS222 solution

    Notes:
    1. Filter solution (0.22 µm-pore filter).
    2. Store at room temperature.
  2. Fixing solution (4% PFA, 1x PBS, 0.1% Tween 20)

    Notes:
    1. Prepare under a fume hood.
    2. Use fresh.
    3. Store at 4 °C.
  3. Sylgard-coated Petri dish
    1. Stir until homogeneous
    2. Degas
    3. Pour about 3 mm into a 60 mm-diameter Petri dish
    4. It will take up to 24 h to cure completely
  4. PBST (1x PBS, 0.01% Tween 20)

    Notes:
    1. Filter solution (0.22 µm-pore filter).
    2. Store at room temperature.
  5. Hydrogel solution

    Notes:
    1. Always make fresh.
    2. Prepare under the fume hood.
    3. Store at 4 °C.
  6. SDS solution (8% SDS, 0.2 M boric acid, pH 8.5)

    Notes:
    1. Filter solution (0.22 µm-pore filter).
    2. Store at room temperature.
  7. Depigmentation
    1. Pre-incubation solution (SSC 0.5x-Tween 20)


    2. Depigmentation solution

      Notes:
      1. Always make fresh.
      2. Prepare under the fume hood.
  8. Storage solution

    Notes:
    1. Store at -20 °C.
    2. Keep at 4 °C after thawing.
  9. Blocking solution

    Notes:
    1. Make aliquots of 10 ml in 15 ml Falcon tubes.
    2. Store at -20 °C.
    3. Keep at 4 °C after thawing.
  10. Staining solution

    Notes:
    1. Make aliquots of 10 ml in 15 ml Falcon tubes.
    2. Store at -20 °C.
    3. Keep at 4 °C after thawing.
  11. Fructose-based high-refractive index solution (fbHRI)
    The fbHRI solution is generated by mixing three solutions prepared separately in advance:
    1. Aqueous diluent with a refractive index (RI) of ~1.334

      Notes:
      1. No need to adjust RI.
      2. Store at 4 °C.
    2. ~90% fructose solution with an RI of 1.457 (F1457)

      1. In a beaker, dissolve 800 g of fructose in ~ 300 ml diluent, mix with a spoon
      2. Cover the fructose solution to prevent evaporation and stir with a strong magnetic stirrer overnight to obtain a homogenous solution. Make up the volume to 800 ml with diluent to obtain a 100% (w/v) fructose solution. Set aside 50 ml of 100% (w/v) fructose for final RI adjustment
      3. Measure RI and adjust the fructose solution to an RI of 1.457 with diluent
    3. ~85% DMSO solution with an RI of 1.457 (DMSO1457)

      Measure RI and adjust the DMSO solution to an RI of 1.457 with diluent
    4. fbHRI solution
      1. Mix F1457 and DMSO1457 in the ratio of 80/20
      2. Measure refractive index with a refractometer
      3. Adjust RI:
        Add 100% fructose to raise RI, add diluent to lower RI
      4. Aliquot into 50 ml Falcon tubes and store at 4 °C
      5. Control and adjust refractive index to 1.457 with a refractometer before use

Acknowledgments

We would like to thank Dr. Spencer Brown for suggesting the use of DiIC18 as a fiducial marker. This work benefited from the facilities and expertise of TEFOR-Investissement d’avenir, and support from the Association Nationale de la Recherche et de la Technologie (ANR-II-INBS-0014). The authors declare no competing financial interests.

References

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  3. Chung, K., Wallace, J., Kim, S. Y., Kalyanasundaram, S., Andalman, A. S., Davidson, T. J., Mirzabekov, J. J., Zalocusky, K. A., Mattis, J., Denisin, A. K., Pak, S., Bernstein, H., Ramakrishnan, C., Grosenick, L., Gradinaru, V. and Deisseroth, K. (2013). Structural and molecular interrogation of intact biological systems. Nature 497(7449): 332-337.
  4. Dambroise, E., Simion, M., Bourquard, T., Bouffard, S., Rizzi, B., Jaszczyszyn, Y., Bourge, M., Affaticati, P., Heuze, A., Jouralet, J., Edouard, J., Brown, S., Thermes, C., Poupon, A., Reiter, E., Sohm, F., Bourrat, F. and Joly, J. S. (2017). Postembryonic fish brain proliferation zones exhibit neuroepithelial-type gene expression profile. Stem Cells 35(6): 1505-1518.
  5. Filippi, A., Mahler, J., Schweitzer, J., Driever, W. J. (2010). Expression of the paralogous tyrosine hydroxylase encoding genes th1 and th2 reveals the full complement of dopaminergic and noradrenergic neurons in zebrafish larval and juvenile brain. J Comp Neurol 518(4): 423-38.
  6. Fini, J. B., Mughal, B. B., Le Mevel, S., Leemans, M., Lettmann, M., Spirhanzlova, P., Affaticati, P., Jenett, A. and Demeneix, B. A. (2017). Human amniotic fluid contaminants alter thyroid hormone signalling and early brain development in Xenopus embryos. Sci Rep 7: 43786.
  7. Frétaud, M., Riviere, L., Job, E., Gay, S., Lareyre, J. J., Joly, J. S., Affaticati, P. and Thermes, V. (2017). High-resolution 3D imaging of whole organ after clearing: taking a new look at the zebrafish testis. Sci Rep 7: 43012.
  8. Ke, M. T., Fujimoto, S. and Imai, T. (2013). SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat Neurosci 16(8): 1154-1161.
  9. Mayrhofer, M., Gourain, V., Reischl, M., Affaticati, P., Jenett, A., Joly, J. S., Benelli, M., Demichelis, F., Poliani, P. L., Sieger, D. and Mione, M. (2017). A novel brain tumour model in zebrafish reveals the role of YAP activation in MAPK- and PI3K-induced malignant growth. Dis Model Mech 10(1): 15-28.
  10. Lawrence, C., Best, J., Cockington, J., Henry, E. C., Hurley, S., James, A., Lapointe, C., Maloney, K. and Sanders, E. (2016). The complete and updated “Rotifer Polyculture Method” for rearing first feeding zebrafish. J Vis Exp (107): e53629.
  11. Lawrence, C. and Mason, T. (2012). Zebrafish housing systems: a review of basic operating principles and considerations for design and functionality. ILAR J 53(2): 179-191.
  12. Oksanen, K. E., Halfpenny, N. J., Sherwood, E., Harjula, S. K., Hammaren, M. M., Ahava, M. J., Pajula, E. T., Lahtinen, M. J., Parikka, M. and Ramet, M. (2013). An adult zebrafish model for preclinical tuberculosis vaccine development. Vaccine 31(45): 5202-5209.
  13. Than-Trong, E. and Bally-Cuif, L. (2015). Radial glia and neural progenitors in the adult zebrafish central nervous system. Glia 63(8): 1406-1428.
  14. Tomer, R., Ye, L., Hsueh, B. and Deisseroth, K. (2014). Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat Protoc 9(7): 1682-1697.
  15. Treweek, J. B., Chan, K. Y., Flytzanis, N. C., Yang, B., Deverman, B. E., Greenbaum, A., Lignell, A., Xiao, C., Cai, L., Ladinsky, M. S., Bjorkman, P. J., Fowlkes, C. C. and Gradinaru, V. (2015). Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. Nat Protoc 10(11): 1860-1896.
  16. Xavier, A. L., Fontaine, R., Bloch, S., Affaticati, P., Jenett, A., Demarque, M., Vernier, P. and Yamamoto, K. (2017). Comparative analysis of monoaminergic cerebrospinal fluid-contacting cells in Osteichthyes (bony vertebrates). J Comp Neurol 525(9): 2265-2283.
  17. Yang, B., Treweek, J. B., Kulkarni, R. P., Deverman, B. E., Chen, C. K., Lubeck, E., Shah, S., Cai, L. and Gradinaru, V. (2014). Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158(4): 945-958.
  18. Parichy DM, Elizondo MR, Mills MG, Gordon TN and Engeszer RE. (2009). Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish. Dev Dyn 238(12): 2975-3015.

简介

在脑功能研究中,了解潜在的神经结构是至关重要的。非常年轻的斑马鱼幼虫被广泛用于神经构造研究,由于它们的大小和自然透明度。然而,这个模型系统有一些局限性,由于使用的动物不成熟,发育率高,行为表现有限。

我们在这里描述了被动清除技术(PACT)的改进版本(Chung等人,2013; Tomer等人,2014; Yang等人, ,2014; Treweek et。,2015),这有助于大型水生物种标本的神经解剖学研究。这种方法最初是为斑马鱼(2017年)开发的( Danio rerio )(2017年;Frétaud等人,2017年; Mayrhofer等人,2017年; Xavier “)等人,2017),但也已经成功地在其他鱼类上进行了测试,如鲭鱼(Oryzias latipes )(Dambroise et al。,2017 ),墨西哥洞穴鱼( Astyanax mexicaus )和非洲斑马鱼(metriaclima zebra )以及其他水生物种,如非洲爪蟾属。 (非洲爪蟾热带非洲爪蟾)(Fini等人,2017)。该协议基于由Deisseroth实验室等开发和修改的CLARITY方法(Chung等人,2013; Tomer等人,2014; Yang等人,2014),适用于水生物种,特别是斑马鱼(zPACT)。

该协议旨在通过在聚丙烯酰胺/甲醛网状物中交联来保持组织的整体结构,使斑马鱼标本光学透明。然后通过SDS处理除去存在于样品中的大部分脂质,通过消除导致不透明的水/脂质界面处的光散射来均化样品的折射率。最后的清除步骤包括将样品在基于果糖的固定介质(衍生自SeeDB)(Ke等人,2013)中孵育,其折射率与物镜的折射率相匹配的显微镜。该技术与使用绿色荧光蛋白(GFP)在特定细胞群体中表达的转基因斑马鱼的组合提供了描述其他技术不可见的解剖细节的机会。

【背景】成年硬骨鱼(斑马鱼,青aka)越来越多地被用作从干细胞到大脑发育的研究中的模型脊椎动物(Than-Trong和Bally-Cuif,2015; Dambroise等人,2017),在生理学和医学研究(Oksanen等人,2013)的所有方面以及对诸如学习,记忆和成瘾等复杂类型的行为的研究(Bailey等人 ,2015)。

作为一个科学服务提供者,Tefor核心设施已经开发了一种可靠的组织清理技术,用于直径数毫米的致密的神经元组织,以满足大型水生动物标本研究的需求。

通过创建一套不同发育阶段的神经解剖学图谱,该协议将作为其他希望将数据整合到这些解剖参考框架(ARF)的实验室的资源。在发表时,7dpf ARF的原型可以从 https://www.zebrafish.tefor.net / 。通过这个URL访问的数据随后将被整合到一个注释的3D神经解剖学图谱中,并且为不同的发育阶段提供标准化的高分辨率共焦成像数据。 Tefor核心设施目前正在开发5dpf,6dpf,7dpf幼虫头和幼年大脑的ARFs。其他阶段ARFs的发展将取决于斑马鱼研究团体成员的需求。

将新样本整合到相应的ARF中需要对样本的发育阶段进行精确的分期以及使用DiI染色,如本文所述。这种染料被用作计算三维成像数据对准ARF的基准标记。但是,如果不需要将生成的数据与现有数据池进行直接比较,则也可以在没有这些步骤的情况下运行该协议。

作为标本大小和成熟度之间的折衷,我们在本协议的发展过程中将重点放在幼年斑马鱼大脑。幼年斑马鱼大脑比较老的鱼小,但可能被认为是成熟的(Filippi,2010)。此外,使用性未成熟的动物应减少由于性二态性导致的数据变异(Ampatzis,2012)。这些动物体积较小也使协议更快,因为它的所有步骤都取决于所应用的药剂渗入标本。然而,所使用的动物已经达到了足够进步的发育阶段,以便将发现推断到较老的标本。

关键字:PACT, 组织清除, 深度成像, 斑马鱼, 免疫组化检查, 共聚焦显微镜

材料和试剂

  1. 无菌50毫升锥形管(康宁,目录号:430291)
  2. 尖锐的刀片(例如剃刀刀片(例如剃刀刀片公司,目录号62-0167)或适当的手术刀刀片(例如,#10) >
  3. 94×16毫米培养皿(Greiner Bio One International,目录号:632102)


  4. 小型刮刀用于在分级过程中操作麻醉的鱼
  5. 无菌15 ml锥形管(Corning,目录号:431470)
  6. 画笔(,例如,Kolinsky France,Manet 630,尺寸3)
  7. 60×15毫米培养皿(Greiner Bio One International,目录号:628160)

  8. 多聚合物防护手套(Shield Scientific,目录号:66 9253)
  9. Parafilm M(Sigma-Aldrich,Parafilm,目录号:P7543)
  10. 用于烤肉炉杂交炉的玻璃管(Fisher Scientific,目录号:11791436)
  11. 2 ml Eppendorf管(STARLAB INTERNATIONAL,目录号:E1420-2000)
  12. 3.2毫升转移吸管(卡尔罗斯,目录号:EA67.1)
  13. 2毫升玻璃管(卡尔罗斯,目录号:H303.1)
  14. 0.22微米孔径的过滤器(SARSTEDT,目录号:83.1826.001)
  15. 标准条件下饲养的6周龄斑马鱼(密度,饲养,等等);劳伦斯和梅森,2012;劳伦斯等人,2016)
  16. 轮虫( brachionus plicatilis )
  17. 盐水虾(无脂肪nauplii )
  18. 干粮(Skretting,Gemma Micro)
  19. 纯氮(液化空气,ALPHAGAZ 1)
  20. Alexa Fluor 488共轭二抗,山羊抗鸡IgY(H + L)(Thermo Fisher Scientific,Invitrogen TM,目录号:A-11039)
  21. (3)(Thermo Fisher Scientific,Invitrogen TM,产品目录号:D282)。
  22. 二甲基亚砜(DMSO)(Carl Roth,目录号:4720.1)
  23. 针对GFP(鸡)的一抗(Aves Lab,目录号:GFP-1020)
  24. 低熔点琼脂糖(Sigma-Aldrich,目录号:A4018)
  25. 矿物油(卡尔罗斯,目录号:HP50.3)
  26. 甲磺酸三卡因(MS222)(Sigma-Aldrich,目录号:A5040)
  27. 碳酸氢钠(卡尔罗斯,目录号:0965.2)
  28. 20x PBS(Santa Cruz Biotechnology,目录号:sc-362183)
  29. 16%甲醛(w / v),无甲醇(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:28908)
  30. 4%甲醛溶液,无甲醇(w / v)(PFA)(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:28908)
  31. 吐温20(Sigma-Aldrich,目录号:P7949)
  32. 在H 2 O(Sigma-Aldrich,目录号:01697)中的40%丙烯酰胺溶液。
  33. VA-044引发剂(Wako Pure Chemical Industries,目录号:017-19362)
  34. SDS颗粒(卡尔罗斯,目录号:CN30.3)
  35. 硼酸(Carl Roth,目录号:6943.1)
  36. 20x SSC(Carl Roth,目录编号:1054.1)
  37. 甲酰胺(CH 3 NO)(Carl Roth,目录号:6749.3)
  38. H 2 O 2溶液(30%)(Carl Roth,目录号:CP26.5)。
  39. 叠氮化钠(Sigma-Aldrich,目录号:S2002)
  40. 正常山羊血清(NGS)(抗体,目录号:54-410)
  41. Triton X-100(Fisher Scientific,目录号:BP151-500)
  42. D( - ) - 果糖(卡尔罗斯,目录号码:4981.6)
  43. SYLGARD 184(Sigma-Aldrich,目录号:761036)
  44. 10倍MS222解决方案(见配方1)
  45. 固定溶液(4%PFA,0.01M PBS,0.1%吐温20)(见配方2)
  46. Sylgard涂层的培养皿(见配方3)
  47. PBST(1×PBS,0.01%吐温20)(见方案4)
  48. 水凝胶溶液(见方法5)
  49. SDS溶液(8%SDS,0.2M硼酸,pH8.5)(见配方6)
  50. 脱色(参见7)
    1. SSC 0.5x-吐温20
    2. 脱色解决方案
  51. 存储解决方案(见方法8)
  52. 阻止解决方案(见第9条)
  53. 染色溶液(见配方10)
  54. 基于果糖的高折射率溶液(fbHRI)(见11)

设备

  1. (例如,Tecniplast 1L育苗池,不含内部部分)
  2. 层压测量网格(见图1)
  3. 通风柜
  4. 冰浴&amp;冰箱
  5. 胚胎勺(卡尔罗斯,目录号:TL85.1)
  6. 杜蒙镊子,直(世界精密仪器,目录号:500336)
  7. 杜蒙镊子,直(世界精密仪器,目录号码:14099)
  8. 杜蒙特镊子,45°角(世界精密仪器,目录号:500234)
  9. 立体显微镜(奥林巴斯,型号:SZX10)
  10. 光纤光源(奥林巴斯,型号:KL 2500 LED)
  11. 加热真空干燥器(VWR,SELECTA,目录号:SELE4000474)
  12. 电炉(Labexchange,Bioblock Scientific,型号:AM3001K)
  13. 真空泵(Savant,型号:Gel Pump GP 110)
  14. 组织学处理盒(Simport,型号:M503-12)
  15. 400毫升烧杯(VWR,目录号:213-1125)
  16. 600毫升烧杯(VWR,目录号:213-1126)
  17. Rotisserie杂交烤箱(Grant Boekel,型号:HIR 1011)
  18. 三维摇杆Polymax 1040(Heidolph Instruments,目录号:036130320)
  19. 折射计(KRUSS Optronic,产品目录号:DR 301-95)
  20. Leica TCS SP8激光扫描显微镜(Leica Microsystems,型号:Leica TCS SP8)
    1. 2个光电倍增管用于同时检测两个通道
    2. Leica HC FLUOTAR L 25x / 1.00 IMM motCorr物镜(Leica Microsystems,产品目录号:15507703)
  21. 重型磁力搅拌棒(SP Scienceware - Bel-Art Products - H-B Instrument,目录编号:F37118-0002)

软件

  1. 斐济/ ImageJ( http://fiji.sc
  2. Amira( https://www.fei.com/software/amira-三维生命科学/
  3. ITK快照( http://www.itksnap.org

程序

  1. 饲养和分期
    在控制条件下(光周期= 14h / 10h光照/黑暗;温度= 26.5±1℃; pH = 7.8±0.1;电导率= 240±30μS/ cm,NH 4 < L = 0mg / L,NO2 = 0mg / L,NO3 <0> 50mg / L),每1.4L罐50个个体组。它们在前两周用轮虫( Brachionus plic a tilis ;> 1,000次/鱼/天;中午一次)然后用卤水(卤虫)和干粮(Skretting,Gemma Micro;明显的饱食,两个剂量,上午和下午)(劳伦斯和梅森,2012; Lawrence等人,2016)。所有程序均按照欧盟指令2010/63 / EU执行。
    正如之前的研究(Parichy等人,2009)所建议的,感兴趣阶段(幼年)是根据鳍褶皱的消失定义的,在大约5-6周当地的标准条件,对应的身体长度为12.5毫米。
    测量和分期步骤在潜在的致死性麻醉和水外进行。因此,必须迅速,单独,非常小心地执行这些步骤,以确保所有的鱼都能够幸存下来。在固定之前应该直接杀死所需大小的鱼,以防止样本中表达模式的衰减。测量/分期应在鱼类设施中进行,所以这些动物必须保持活性,直到它们被转移到实验室并在通风橱下杀死和固定。太小的鱼可以放置几天,然后重新测量,太大的鱼可以引入其他实验或育种计划。
    1. 准备麻醉溶液:200毫升的1x MS222溶液,通过在鱼缸(麻醉池)中的鱼水中稀释10倍的MS222溶液(见配方1)1:9。
    2. 对于每条鱼线,准备两个额外的鱼缸:
      1. 鱼线的名称+正确的尺寸。
      2. 鱼线名称+大小不正确。

    3. 每次麻醉一只鱼,将其转移到麻醉罐中。
      等到鱼完全麻醉后通过用刮刀触摸尾部并检查运动来测试这一点。
    4. 将鱼放置在测量网格上(图1)。
      1. 去除鱼周围的液体,以避免镜头效应。
      2. 使用刮刀,将鱼的嘴部对准灰色的中线,并沿水平线将背部定位,如图1所示。


        图1.按年龄分类幼鱼并测量其体长:在测量栅格上的鱼的图表。不同的颜色线对应于距测量栅格中心线的不同距离:内部灰线11 mm;洋红色线12毫米;青线12.5毫米;黄线13毫米;外灰线14毫米。该图中的鱼将被认为具有12.5mm的体长(青线的中心灰线)。斑马鱼图像由数据库生命科学中心(DBCLS)维护的多哥图片库修改。

    5. 测量从嘴巴到尾巴开始的距离。
      注:少年斑马鱼通常测量12.5毫米。
    6. 将鱼放入与其大小相对应的罐中。

      1. 过小的鱼可以收获
      2. 太大的鱼可以用于后期或繁殖/库存维护。
    7. 检查鱼是否完全从麻醉中恢复。
      3-5分钟后,鱼应再次正常游泳。游泳起初可能会比较迟缓,但鱼类应在5-10分钟内完全恢复。

  2. 斑马鱼脑解剖
    1. 准备标本和解决方案
      1. 在通风橱下,准备固定液(4%PFA,1x PBS,0.1%吐温20,见配方2)。在50毫升锥形管中准备40毫升固定溶液,作为5个鱼头的前缀。保持在4°C管。
      2. 按照当地法规( eg ,欧盟指令2010/63 / EU,根据卡尔斯鲁厄理工学院的建议开发的斑马鱼的人道杀害)。
    2. 磁头的前缀
      1. 将样本转移到1x PBS中,并在鳃后面切割头部(保持鳃盖完好),用锋利的刀片以避免任何压缩或中断CNS。
      2. 将头部置于含有固定溶液(用镊子转移)的50ml锥形管中。

      3. 4°C孵育18小时
    3. 解剖
      1. 在通风橱下,准备新的固定溶液。在5毫升锥形管中准备40毫升的固定溶液5鱼的大脑。保持在4°C管。
      2. 使用镊子将预先固定的头转移到填充有PBST的SYLGARD涂覆的培养皿(参见配方3)(参见配方4)。
      3. 在立体显微镜下,用精密的镊子从头部小心提取脑。这一步需要精细的运动技能,可能需要一些训练和实践来发展一个令人满意的解剖技术。 (请参阅补充视频

      4. 用超细镊子从大脑中取出周围组织 注意:
        1. 使用结缔组织作为手柄,避免用镊子触摸大脑。










      5. 使用胚胎勺将解剖的大脑转移到含有固定溶液的50ml锥形管中。
      6. 在4°C孵育5小时。
      7. 继续水凝胶孵化的下一步,或将大脑保存在储存溶液中。
    注:关于解剖过程的详细描述,我们提供了一个解剖电影(见 补充视频 )。

  3. 组织清理
    1. 在水凝胶溶液中孵育
      1. 在通风橱下,在冰上制备水凝胶溶液(参见配方5)。
        注意:水凝胶溶液必须在样品转移到溶液前立即进行新鲜处理。
      2. 在整个实验过程中将样品保存在冰上。
      3. 将样品转移到带有精细油漆刷或切割移液管的15ml Falcon管中。
        最大:8个大脑/管
      4. 从15毫升管中尽可能多地取出液体。
      5. 加入14毫升水凝胶溶液到含有样品的15毫升试管中。

        在4°C孵育48小时。&nbsp;
    2. 聚合
      1. 在通风橱下,设置干燥器,并将电炉置于37°C。

      2. 标记一个60毫米的陪替氏培养皿,其中包含待处理样本的名称和编号
      3. 将培养皿放在37°C的电炉上。

      4. 所有14毫升包含大脑的水凝胶溶液倒入培养皿。
      5. 保持培养皿敞开和发现。
      6. 关闭干燥器,确保密封。
      7. 用真空泵除去空气,直到压力表读数为-50厘米汞柱。
      8. 仔细重新填充纯氮气,直到压力表读数为0.
      9. 重复最后两步6次。
      10. 让样品聚合2.5小时。
    3. 在SDS解决方案中被动清除
      1. 大脑聚合后按以下步骤操作:
        1. 将干净的盒子放在含有8%SDS溶液的60毫米培养皿中(见6)。
        2. 佩戴多聚物防护手套,使用弯曲的镊子从聚合的水凝胶中分别去除每个大脑,并将其放在手中。
        3. 使用细软的油漆刷和8%SDS溶液,轻轻地清洁大脑,除去所有周围的水凝胶。
        4. 将大脑放入卡带。
        5. 关闭磁带并将它们放入盛有8%SDS溶液的300毫升烧杯中。
      2. 用Parafilm盖上烧杯,在室温下避光保存2-5小时,透析剩余的PFA,引发剂和单体。
      3. 将盒子放入含有8%SDS溶液的高压灭菌的旋转式杂交烘箱玻璃管中。

      4. 在杂交培养箱中37°C旋转孵育杂交管
      5. 每天检查样品的透明度(图2)。
        注意:青少年大脑的标准清理时间为8天。
      6. 一旦样品成功清除(图2),在PBST中清洗三次。每天更换洗涤液一次,连续两天。
      7. 如果样品在洗涤第三天后不立即使用,请将其储存在4°C的储存溶液中(见配方8)。


        图2.组织清除过程的时间序列:在8天内实现幼年斑马鱼大脑的被动清除(如步骤C3c-C3e中所述)。该图说明了8%SDS中的幼鱼脑部的清除过程随时间的推移,显示透明度的增加来自:A.第0天,第2天,第6天,第D天8.比例尺= 1毫米。

  4. 色素脱失
    1. 在60毫米的培养皿中倒入10毫升的预孵育液(SSC 0.5x-吐温20,见方法7),并用油漆刷或胚胎汤匙将样品转移到培养皿中。
      注意:确保用PBST稀释预孵育溶液。
    2. 在室温下孵育1小时。
    3. 在通风橱下,使用前立即准备脱色素溶液(参见配方7)。
    4. 再次在通风橱下,用一些液体取出样品(以确保它们不会变干),并将它们放在培养皿的盖子上。从盘中取出所有的预孵育溶液,并用脱色溶液代替。用最少的预孵育溶液将样品返回到培养皿中。
      注意:保持培养皿裸露在光下加速脱色过程。
    5. 每10分钟检查一次脱色的进展情况。在立体显微镜下检查色素沉着进展时,盖上培养皿。
      注:少年大脑的标准脱色时间为45分钟。
    6. 一旦脱色完成后,在通风橱下用PBST冲洗两次。
      注意:在进行下一步之前,将冲洗过的样品放在工作台上过夜或至少4小时。

  5. 后固定
    1. 将样品放入2毫升Eppendorf管中。去除所有的液体,并添加1毫升的定影液。
    2. 在4°C孵育过夜。
      注意:
      1. 如果在此步骤后立即使用样品,则在PBST中冲洗4次3小时。
      2. 如果样品不能立即使用,请取出定影液并更换为1 ml的存储溶液。将样品储存在4°C直到使用。

  6. 整装标签
    1. 将样品转移到带有切割移液管的2ml玻璃管中。最大:5个大脑/管。
    2. 为了防止稀释下一个溶液,尽可能多地从管中取出液体。
    3. 加入1毫升封闭液(见配方9)到每个管。
    4. 在4°C孵育过夜。
    5. 去除封闭液,每管加600μl染色液(见10)。
    6. 每管加入1.2μl抗GFP抗体,得到1/500稀释液。
      注:其他一抗已成功测试。但是必须优化稀释度,一抗浓度一般在1μg/ ml到10μg/ ml之间。

    7. 在室温下孵育7天,轻轻搅动,在3D摇摆器上
    8. 在PBST中冲洗3次,每次20分钟。
    9. 从管中取出所有液体,每管加入600μl染色液。

    10. 每个试管中加入1.5μlAlexa Fluor 488结合的第二抗体,以获得1/400的稀释度

    11. 在室温下孵育5天,轻轻摇动,在3D摇杆上
    12. 在PBST的几个变化中冲洗2天。
      1. 可选:将DiI染色用作基准标记。
        孵育样品2天,每管600μL染色液,稀释1μM。
        注意:通过将DiI粉末溶解在DMSO中制备1mM Dil储备溶液。
      2. 用PBST冲洗DiI染色的样品最少3小时。

  7. 安装和成像
    1. 准备成像
      1. 将样品在50%fbHRI / 50%稀释液中孵育至少6小时(见11和图3B)
      2. 在fbHRI中孵育至少12小时(图3C)。
    2. 为了成像,将样品以60%培养皿中的1%(wt / vol)低熔点琼脂糖液滴加,并用fbHRI将其大量覆盖(深度约10mm)。
    3. 为了避免在长时间的成像过程中蒸发,覆盖一层矿物油。
    4. 使用配备有Leica HC FLUOTAR L 25x / 1.00 IMM motCorr物镜的Leica TCS SP8激光扫描共聚焦显微镜可以对整个装载的脑荧光进行成像。通过用波长为488和552nm的激光以1-3%的激光功率激发荧光团来检测荧光信号,并通过两个内部光电倍增器(PMT)捕获。需要拼接扫描成像,然后马赛克拼接才能捕捉整个大脑的图像。


      图3.通过均质化果糖基高折射率介质(fbHRI)中的折射率,被动清除后幼年斑马鱼脑的最终清除。 :一种。用PBS替代被动清除过程的SDS溶液后,组织再次变得不透明。 B.在PBS中的50%fbHRI中温育后,不透明度显着降低。 C.在100%fbHRI中孵育后,组织清除过程完成。比例尺= 1毫米。

数据分析

成像数据的分析在很大程度上取决于科学问题。因此有许多方法可用。 Dambroise等人(2017年)使用这里描述的组织清除技术来显示斐济完整标本部分的最大强度投影。在ITK-snap或Amira(FEI)中可以实现手动和机器辅助的表达模式分割以及所得区域的可视化。典型的用这里描述的组织澄清技术治疗的大样本可以分析,如视频1所示,一个翻译电影,其中原始堆栈的每一片被转换成电影帧。标准的mp4压缩减少了几个数量级的文件大小,方便访问。

视频1

食谱

  1. 10倍MS222解决方案

    注意:
    1. 过滤解决方案(0.22微米孔过滤器)。
    2. 在室温下储存。
  2. 定影液(4%PFA,1x PBS,0.1%吐温20)

    注意:
    1. 准备在通风橱下。
    2. 使用新鲜的
    3. <4>保存在4°C。
  3. Sylgard涂层培养皿
    1. 搅拌直至均匀
    2. 德加
    3. 向直径60毫米的培养皿中倒入约3毫米

    4. 完全治愈需要24小时
  4. PBST(1x PBS,0.01%吐温20)

    注意:
    1. 过滤解决方案(0.22微米孔过滤器)。
    2. 在室温下储存。
  5. 水凝胶溶液

    注意:
    1. 始终保持新鲜感。
    2. 准备在通风橱下。
    3. <4>保存在4°C。
  6. SDS溶液(8%SDS,0.2M硼酸,pH8.5)

    注意:
    1. 过滤解决方案(0.22微米孔过滤器)。
    2. 在室温下储存。
  7. 色素脱失
    1. 预孵育液(SSC 0.5x-Tween 20)


    2. 脱色解决方案

      注意:
      1. 始终保持新鲜感。
      2. 准备在通风橱下。
  8. 存储解决方案
    “”
    注意:
    1. 储存在-20°C。
    2. 解冻后保持在4°C。
  9. 阻止解决方案

    注意:
    1. 在15毫升Falcon管中制成10毫升等分试样。
    2. 储存在-20°C。
    3. 解冻后保持在4°C。
  10. 染色解决方案

    注意:
    1. 在15毫升Falcon管中制成10毫升等分试样。
    2. 储存在-20°C。
    3. 解冻后保持在4°C。
  11. 基于果糖的高折射率溶液(fbHRI)
    fbHRI解决方案是通过混合三种预先单独制备的解决方案来生成的:
    1. 折射率(RI)为〜1.334的水性稀释剂
      注意:
      1. 无需调整RI。
      2. <4>保存在4°C。
    2. 〜90%果糖溶液,RI为1.457(F1457)

      1. 在烧杯中,将800g果糖溶解在〜300ml稀释液中,用勺子混合
      2. 盖上果糖溶液以防止蒸发,并用强磁力搅拌器搅拌过夜以获得均匀的溶液。用稀释液补足体积至800ml,得到100%(w / v)的果糖溶液。撇开50毫升的100%(w / v)果糖用于最终的RI调整
      3. 测量RI,用稀释液将果糖溶液调整到1.457的RI
    3. 〜85%DMSO溶液,RI = 1.457(DMSO1457)

      测量RI,用稀释剂将DMSO溶液调整到1.457的RI
    4. fbHRI解决方案
      1. 以80/20
        的比例混合F1457和DMSO1457
      2. 用折射计测量折光指数
      3. 调整RI:
        加入100%果糖升高RI,加入稀释剂降低RI
      4. 分装到50毫升Falcon管中,并在4°C储存
      5. 使用前用折光仪控制和调整折射率为1.457

致谢

我们要感谢斯宾塞布朗博士建议使用DiIC18作为基准标记。这项工作得益于TEFOR-Investissement d'avenir的设施和专业知识以及国家研究与技术协会(ANR-II-INBS-0014)的支持。作者声明没有竞争的财务利益。

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引用:Affaticati, P., Simion, M., De Job, E., Rivière, L., Hermel, J., Machado, E., Joly, J. and Jenett, A. (2017). zPACT: Tissue Clearing and Immunohistochemistry on Juvenile Zebrafish Brain. Bio-protocol 7(23): e2636. DOI: 10.21769/BioProtoc.2636.
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