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Feb 2019

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Retina Injury and Retina Tissue Preparation to Study Regeneration in Zebrafish
通过斑马鱼视网膜损伤和视网膜组织制备进行视力恢复研究   

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Abstract

Unlike mammals, primitive vertebrates have immense capability to regenerate almost all of their organs including the central nervous system. Among primitive organisms, zebrafish have been extensively used as a model system for regeneration studies. The retina is a part of the central nervous system and mammals lack the potential to repair any damage caused to it. Zebrafish have been used for retina regeneration studies because of ease in handling and maintenance. In zebrafish, Muller glia cells respond to damage and enter the regenerative cascade to maintain the retinal homeostasis. Zebrafish retinal damage can be induced by light, chemical or mechanical methods. Here we are describing the mechanical method of retinal injury, which ensures uniform damage to all retinal layers. Alongside this, we have also described in vivo manipulation strategies for the regeneration associated genes and preparation of retinal tissue for immunohistochemical analysis.

Keywords: Zebrafish (斑马鱼), Retina (视网膜), Electroporation (电穿孔), Intravitreal (玻璃体内), Transfection (转染), Eye (眼睛)

Background

Retina is the sensory part of the eye and any physical or physiological damage leads to impairment of vision. In the course of evolution higher vertebrates have lost the regenerative potential while primitive vertebrates have enormous capability to regenerate their lost vision. Studying the regenerative events in primitive organisms such as zebrafish can be a hope for mammalian regeneration studies. Zebrafish have been used for retina regeneration studies with various injury paradigms being used. The photobleaching method damages the photoreceptor cells, while chemical methods damage the ganglion cell layer. Mechanical injury by the stab wound method maintains a uniform injury to all retinal layers. Various approaches including transgenic approaches have been used to find the relevance of genetic events during retina regeneration. Here we are describing the in vivo method for mRNA transfection which allows manipulation of gene expression levels above endogenous levels.

Materials and Reagents

  1. 30G needle (BD Microlance, 30G ½”, 0.3 x 13 mm, catalog number: 304000)
  2. Centrifuge tubes (Tarsons, catalog number: 500010x)
  3. Paper towels (Scott SCOTTFOLD Towels, 31.4 cm x 19.9 cm, catalog number: 01960)
  4. Hamilton syringe (Hamilton Company, 10 µl, catalog number: MICROLITERTM #701)
  5. Staining box (Custom made by fixing two plastic pipettes parallel to each other at the bottom of a rectangular plastic box)
  6. Glass slides (Superfrost Plus Microscope Slides) (Fisher Scientific, catalog number: 12-550-15)
  7. pCS2+ plasmid (David Turner, University of Michigan, Ann Arbor)
  8. Ethyl 3-aminobenzoate, methane sulfonic acid salt, Tricaine methanesulfonate (Acros, catalog number: 118000500)
  9. 2x HBSS (Diluted from 10x solution, see Recipes)
  10. mMESSAGE mMACHINE SP6 kit (Thermo Fisher, catalog number: AM1340)
  11. Lipofectamine messenger max reagent (Invitrogen, catalog number: LMRNA001)
  12. DABCO (1,4 diazabicyclo [2.2.2] octane, Sigma-Aldrich, catalog number: D27802)
  13. Na2HPO4 (Sodium phosphate dibasic, Sigma-Aldrich, catalog number: 255793)
  14. NaH2PO4 (Sodium phosphate monobasic, Sigma-Aldrich, catalog number: 33198)
  15. KCl (Potassium chloride, Himedia, catalog number: MB043)
  16. HEPES (Sigma-Aldrich, catalog number: H3375)
  17. Glucose (Sigma-Aldrich, catalog number: G8270)
  18. Tissue-Plus O.C.T. compound (Fisher Health care, catalog number: 4585)
  19. BSA (Bovine serum albumin fraction-V, Himedia, catalog number: GRM105)
  20. Triton X-100 (Sigma-Aldrich, catalog number: T8787)
  21. BrdU (5-Bromo-2’-deoxyuridine, Sigma-Aldrich, catalog number: B5002)
  22. PFA (Paraformaldehyde, Sigma-Aldrich, catalog number: P6148)
  23. PVA (Polyvinyl alcohol, Sigma-Aldrich, catalog number: P8136)
  24. Glycerol (Sigma-Aldrich, catalog number: G7757)
  25. Tris Base (Trizma base, Sigma-Aldrich, catalog number: T1503)
  26. HCl (Hydrochloric acid, Sigma-Aldrich, catalog number: 320331)
  27. Sucrose (Sigma-Aldrich, catalog number: S0389)
  28. Tris-HCl (1 M, pH 7.5, 100 ml) (see Recipes)
  29. Tricaine methanesulfonate solution (100 ml) (see Recipes)
  30. Phosphate buffer (PB, 1 M, pH 7.4, 100 ml) (see Recipes)
  31. Phosphate buffered saline (PBS, 10x, pH 7.4, 1 L) (see Recipes)
  32. HBSS (Hanks balanced salt solution, 10x, pH 7.14, 100 ml) (see Recipes)
  33. 4% PFA (50 ml) (see Recipes)
  34. DABCO (2.5%, 50 ml) (see Recipes)
  35. Sucrose solutions (50 ml) (see Recipes)

Equipment

  1. Forceps (McPherson Suture Tying Forceps Straight-With Tying Platform 10cm (4”) 5 mm size, jaw length, Surtex Instruments, catalog number: FR-780-10)
  2. Stereomicroscope (Carl ZeissTMStemiTM DV4 Series Stereomicroscopes with LED Illumination)
  3. Electroporator (Electro Square Porator, BTX Harvard Apparatus, model: ECM 830)
  4. Electrodes (Platinum Tweezertrode, 5 mm Diameter with 45-0204 cables, BTX, catalog number: 45-0489)
  5. Cryostat (Leica, model: CM3050 S)
  6. Rotospin Rotary Mixer (Tarsons)
  7. Shaking water bath (Stuart, model: SBS40)
  8. Centrifuge (Labocene, catalog number: ScanSpeed 1736R)
  9. Nikon Ni-E fluorescence microscope and Nikon A1 confocal imaging system (Nikon A1-SHS, catalog number: 10225)

Procedure

  1. Retina Injury (Kaur et al., 2018; Mitra et al., 2018; Mitra et al., 2019; Sharma et al., 2019)
    1. Prepare 50 ml of 1x tricaine methane sulfonate solution in the fish water.
    2. Anesthetize the fish by putting in tricaine solution till the gill’s movement slows down.
    3. Keep the anesthetized fish on a wet paper towel bedding with the right side facing upward.
    4. Focus the zebrafish eye under a stereomicroscope and tilt the dorsal side of eyeball gently with forceps.
    5. Poke one edge of the ventral side of the eye with 30 G needle by stab wound injury and then injure the other edge of the eye.
    6. Repeat the injuries on the dorsal side of the eye by tilting it gently from the ventral side so that a total of four injuries are made.
    7. Let the fish revive in the system water (see Video 1 for details).

      Video 1. Retinal injury in zebrafish

  2. In vivo mRNA transfection (Mitra et al., 2018; Sharma et al., 2019)
    In vivo overexpression of genes is achieved by mRNA transfection in the retina. It involves the following steps (see Video 2 for details):

    Video 2. Intravitreal injection and morpholino electroporation

    1. Preparation of transfection reagent
      1. Clone GFP or gene of interest into pCS2+ plasmid. Linearize the vector containing insert from the 3’ end. Prepare mRNA with a sp6 message machine in vitro transcription kit. Precipitate mRNA and dissolve in nuclease-free water to make a stock of 2,000 ng/µl.
      2. Prepare transfection mixture (8 µl) with lipofectamine 2,000 transfection reagent as follows:
        1. Mix 2 µl each of mRNA (2,000 ng/µl) and 2x HBSS solution at room temperature.
        2. Mix 2 µl of lipofectamine with an equal volume of 2x HBSS solution at a ratio of 1:1 and keep the solution at room temperature.
        3. Let both the solutions stand at room temperature for five minutes.
        4. Mix the solutions (i) and (ii), dropwise at a ratio of 1:1 and keep the mixture the room temperature for 30 min. Use the final transfection mixture containing 500 ng/µl of mRNA for intravitreal injection.
      Note: This transfection mixture is for intravitreal injections in 4-5 zebrafish and only a single concentration of mRNA has been used. Depending upon the experimental need, these volumes can be scaled up or down, without any loss in transfection efficiency.
    2. In vivo overexpression by intravitreal injection
      1. Anesthetize the fish and injure the right eye by making four stab wounds with a 30 G needle.
      2. From the fourth poke inject 1 µl of transfection mixture using Hamilton syringe.
      3. Electroporate the fish by placing a negative electrode on the right eye and positive electrode on the other side.
      4. Electroporate with five pulses at 70 V of 50 milliseconds duration with a gap period of 950 milliseconds between the pulses.
      5. Harvest the eyes on 4 days post-injury as described in sections E and F.
      6. In gene overexpression retina check the proliferation in terms of BrdU positive cells in comparison to GFP transfected retinae (see Procedures E, F and G).
      7. Check the transfection by immunostaining for GFP and subsequent imaging which shows GFP in the whole retina. Two representative images to show GFP expression at the injury site and away from the injury site are appended here for reference (Figure 1).


        Figure 1. Showing GFP expression in zebrafish retina after GFP mRNA transfection. Asterisk marks the injury spot. ONL−outer nuclear layer; OPL−outer plexiform layer; INL−inner nuclear layer; IPL−inner plexiform layer; GCL−ganglion cell layer.

  3. Drug delivery and Morpholino electroporation (Kaur et al., 2018; Mitra et al., 2018; Mitra et al., 2019; Sharma et al., 2019)
    To study the localized effect of drugs, proteins, and gene knockdown approaches, intravitreal injections are made.
    1. Preparation of solution for injection:
      1. Dissolve the lyophilized morpholino (300 ng) in 300 µl of autoclaved Milli-Q water to make a stock of 1 mM. Use neat for 1 mM or dilute with Milli-Q water to make stocks of 0.5 or 0.25 mM for injection.
      2. Dissolve the protein in the recommended solvent to make a stock solution and dilute further in 1x PBS or recommended diluent.
      3. Dissolve the drugs in 1 ml DMSO (Dimethyl sulfoxide) or recommended solvent to make a stock solution of final concentration depending upon the molecular mass and weight of the pharmacological inhibitor. Make the required working concentrations from the main stock by diluting with Milli-Q water.
    2. Injection of the solution:
      1. Injure one retina by stab wound injury as described above.
      2. From the fourth poke inject around 1 µl of the reagent with Hamilton syringe.
      3. In the case of morpholino delivery, electroporate the morpholino to make their entry in the retina.
      4. Place the positive electrode on the eye injected with morpholino and the negative electrode on the other eye.
      5. Electroporate at the conditions given above.
      6. Harvest the eyes on 4 days post retinal injury (dpi) as described below.

  4. BrdU labeling
    BrdU pulse labeling is done for 3 h before harvesting the eye on 4 dpi. It labels actively proliferating cells by incorporating thymidine analog (BrdU) in the replicating DNA.
    1. Make 5 mM solution of BrdU by dissolving 15.35 mg of BrdU powder in 10ml of autoclaved Milli-Q water. Make 1 ml aliquots and store at -20 °C for later use.
    2. Bring the BrdU solution at room temperature before use and fill in the insulin syringe.
    3. Anesthetize the fish and hold on a wet paper towel under the microscope.
    4. Insert the insulin needle gently in the midway between pelvic fins and making needle parallel to the fish body taking care not to damage internal organs.
    5. Inject around one unit (15-20 µl) of BrdU solution and leave the fish for 3 h in the system water.

  5. Harvesting and tissue preparation
    1. Harvesting of eye (see Video 3 for details)
      1. Euthanize the fish by tricaine overdosing (prolonged immersion in 1x tricaine solution).
      2. Gently pull out the eye out of the socket with forceps.
      3. Keep the eye in chilled fixative in the Petri plate and focus under a stereomicroscope.
      4. Hold the eye with forceps and pierce the cornea with a needle. With the help of forceps tear the cornea and gently push the eye allowing the lens come out.

      Video 3. Zebrafish euthanasia and harvesting of the eye

    2. Tissue fixation
      1. Fix the eye overnight at 4 °C in 4% PFA with continuous rotation at 12 rpm on rotor spin.
      2. Remove the PFA and dehydrate the tissue in the sucrose gradient for 45 min each at room temperature with continuous rotation. Add the sucrose solutions as follows:
        5% sucrose, 1 volume
        5%:20% sucrose, 2:1 volume
        5%:20% sucrose, 1:1 volume
        5%:20% sucrose, 1:2 volume
        20% sucrose, 1 volume
      3. Finally, replace 20% sucrose with 500 µl of fresh sucrose solution and add an equal volume of OCT into it. Mix by rotation at room temperature for 30 min.
      4. Make cubic molds of aluminum foil by wrapping on cubits.
      5. Fill the mold till half with OCT, put a labeled flag at the top and place the eye dorsoventrally parallel to it.
      6. Freeze the positioned eye immediately by keeping at -80 °C.

  6. Cryo-sectioning and Immunostaining
    1. Cryo-sectioning
      1. Turn on the cryostat and keep eye blocks inside the chamber for 10 min.
      2. Fix the tissue on specimen disc with the help of OCT.
      3. Fix the specimen disc on the specimen head and label the glass slides.
      4. Remove the excess of OCT till retinal tissue starts appearing.
      5. Take 8-10 µm thin serial sections on glass slides.
      6. Allow the slides dry overnight at room temperature.
    2. Immunostaining
      1. Take one set of slides and lay them horizontally on the immunostaining rack.
      2. Give three washes with 1x PBS for 10 min each to remove OCT.
      3. Block the tissues with 5% BSA in PBST (1x PBS with 0.01% Triton X-100) for 2-3 h at room temperature.
      4. Remove the blocking solution and overlay the slides with the primary antibody at 4 °C overnight.
      5. Next day collect the primary antibody and wash the slides three times with PBST at room temperature for 10 min each.
      6. Overlay the slides with fluorescently labeled secondary antibody for 2-3 h at room temperature.
      7. Collect the secondary antibody and wash the slides thrice with PBST.
      8. Stop the reaction by washing with autoclaved water.
      9. Let the slides dry vertically in the dark for 10-15 min.
      10. Mount the slides with 60 µl DABCO and coverslip them.
      11. Let the slides dry overnight at room temperature in a dark chamber.

  7. Image acquisition
    Examine the slides under a fluorescence microscope and take images with a confocal imaging system with a Nikon Ni-E fluorescence microscope equipped with fluorescence optics and Nikon A1 confocal imaging system.

Data analysis

Count the BrdU labeled cells under a fluorescence microscope by observation of their fluorescence in retinal sections. In both experimental and treatment sets count the cells at the main injury spot and avoid the spots away from the main injury. The injury spot can be demarcated by discontinuous outer nuclear layer as shown below (Figure 2). Take a minimum of three eyes in each experimental setup. Count the labeled cells in all the main injury spots present in the three retinal sections. Exclude the outliers (extremely low and extremely high values) and calculate the average and standard deviation by analyzing the data in an Excel sheet.


Figure 2. Showing injury spot (demarcated by an asterisk) with BrdU positive cells labeled in red. Square around the injury spot marking the area for cell count. Scale bar−10 µm; ONL−outer nuclear layer; INL−inner nuclear layer; GCL−ganglion cell layer.

Recipes

  1. Tris-HCl (1 M, pH 7.5, 100 ml)
    1. Make 1 M stock of Tris-HCl by dissolving 12.1 g of Tris base in 80 ml of Milli-Q water
    2. Adjust the pH to 7.5 with concentrated HCl solution and make the final volume to 100 ml with Milli-Q water
  2. Tricaine methane sulfonate solution (100 ml)
    1. Measure 2 g of Ethyl 3-aminobenzoate, methane sulfonic acid salt and dissolve it in 100 ml of 0.1 M Tris-HCl (pH 7.5) solution (Dilute the stock solution to 0.1 M by taking 10 ml of 1 M solution in 90 ml of Milli-Q water)
    2. Keep the solution at 4 °C and dilute it (1:100) in fish system water before use
  3. Phosphate buffer (PB, 1 M, pH 7.4, 100 ml)
    1. Weigh the following components:
      Na2HPO4
      10.9877 g
      NaH2PO4
      2.711548 g
    2. First, dissolve Na2HPO4 in 80 ml of Milli-Q H2O, then add NaH2PO4
    3. Adjust the pH to 7.4 and make up the volume to 100 ml with Milli-Q H2O
    4. Autoclave the solution and dilute to a working stock of 0.1 M with autoclaved Milli-Q water
  4. Phosphate buffered saline (PBS, 10x, pH 7.4, 1 L)
    1. Measure the following components:
      NaCl
      75.97 g
      Na2HPO4
      9.937 g
      NaH2PO4
      3.59 g
    2. Dissolve all the components in 800 ml of Milli-Q water by vigorous shaking or with magnetic beads
    3. Once all the components have dissolved adjust the pH to 7.4 and make up the final volume to 1,000 ml
    4. Autoclave the solution and use it as a 1x solution diluted with Milli-Q water
  5. HBSS (Hanks balanced salt solution, 10x, pH 7.14, 100 ml)
    NaCl
    8 g
    KCl
    0.354 g
    Glucose
    1.351 g
    HEPES
    5.466 g
    Na2HPO4
    99.35 mg
    1. Measure these components and dissolve in 80 ml of Milli-Q water
    2. Adjust the pH to 7.14 and make up the final volume to 100 ml
    3. Prepare 2x solution from 10x stock and store in 1 ml aliquots at -20 °C
  6. 4% PFA (50 ml)
    1. Measure 2 g of PFA in a 50 ml dry centrifuge tube and add 5 ml of 1 M PB into it
    2. Make the volume to 45 ml with autoclaved Milli-Q water and dissolve PFA by heating at 65 °C in a shaking water bath
    3. Take out the centrifuge tube in-between to mix settled powder by inverting vigorously. It takes around 15-30 min to dissolve completely
    4. After dissolving properly, adjust the volume to 50 ml with autoclaved Milli-Q water and keep immediately on ice. For long term storage, make small aliquots and store at -20 °C
  7. DABCO (2.5%, 50 ml)
    1. Measure 12 g of glycerol in a 50 ml centrifuge tube
    2. Measure 4.8 g of PVA and add it to glycerol. Mix well by gently inverting the tube till homogenous solution
    3. Add 12 ml of autoclaved Milli-Q water and mix overnight on rotor. Then spin at room temperature
    4. Add 24 ml of 0.2 M Tris-HCl, pH 8-8.5
      1. Make 100 ml of 1 M Tris-HCl solution by dissolving 12.1 g of Tris base in 80 ml of Milli-Q water
      2. Adjust the pH to 8.2 with a concentrated HCl solution and make up the final volume to 100 ml
      3. To make 0.2 M Tris-HCl take 6 ml of 1 M stock in 24 ml of Milli-Q water
    5. Heat the solution to 50 °C in a shaker water bath for 15-30 min and centrifuge at 5,000 x g for 15 min
    6. Carefully remove the supernatant and add 1.25 g of DABCO into the supernatant
    7. Centrifuge at 5,000 x g for 15 min
    8. Aliquot the supernatant in 1 ml tubes and store the at -20 °C for later use
  8. Sucrose solutions (50 ml)
    1. 5% sucrose: Measure 2.5 g of sucrose and dissolve it in 40 ml of 0.1 M PB solution. Adjust the volume to 50 ml with 0.1 M PB
    2. 20% sucrose: Measure 10 g of sucrose and dissolve it in 40 ml of 0.1 M PB solution. After dissolving it by rotation, adjust the volume to 50 ml with 0.1 M PB
    3. Store the solutions at 4 °C for long term use

Acknowledgments

P.S acknowledges postdoctoral fellowship support from Wellcome Trust/DBT India Alliance and IISER Mohali. P.S thanks Ajay Kumar, Undergraduate student, for video capturing and editing. This work was supported by the Wellcome Trust/DBT India Alliance Intermediate Fellowship (IA/I/12/2/500630) awarded to Rajesh Ramachandran. R.R also acknowledges research funding from Science Education and Research Board (SERB), DST, India (EMR/2017/001816), DBT India (BT/PR9407/BRB/10/12612013), (BT/PR17912/ MED/31/336/2016) and support from IISER Mohali.

Competing interests

The authors declare that they have no conflict of interest.

References

  1. Kaur, S., Gupta, S., Chaudhary, M., Khursheed, M. A., Mitra, S., Kurup, A. J. and Ramachandran, R. (2018). let-7 microRNA-mediated regulation of shh signaling and the gene regulatory network is essential for retina regeneration. Cell Rep 23(5): 1409-1423.
  2. Mitra, S., Sharma, P., Kaur, S., Khursheed, M.A., Gupta, S., Ahuja, R., Kurup, A.J., Chaudhary, M., and Ramachandran, R. (2018). Histone deacetylase-mediated Muller glia reprogramming through her4.1-lin28a axis is essential for retina regeneration in zebrafish. iScience 7: 68-84.
  3. Mitra, S., Sharma, P., Kaur, S., Khursheed, M.A., Gupta, S., Chaudhary, M., Kurup, A.J., and Ramachandran, R. (2019). Dual regulation of lin28a by Myc is necessary during zebrafish retina regeneration. J Cell Biol 218: 489-507.
  4. Sharma, P., Gupta, S., Chaudhary, M., Mitra, S., Chawla, B., Khursheed, M. A. and Ramachandran, R. (2019). Oct4 mediates Muller glia reprogramming and cell cycle exit during retina regeneration in zebrafish. Life Sci Alliance 2(5): pii: e201900548.

简介

与哺乳动物不同,原始脊椎动物具有巨大的能力来再生几乎所有器官,包括中枢神经系统。在原始生物中,斑马鱼已被广泛用作再生研究的模型系统。视网膜是中枢神经系统的一部分,哺乳动物缺乏修复对其造成的任何损害的潜力。斑马鱼由于易于处理和维护而被用于视网膜再生研究。在斑马鱼中,穆勒神经胶质细胞对损伤做出反应并进入再生级联反应,以维持视网膜稳态。斑马鱼的视网膜损伤可以通过光,化学或机械方法引起。在这里,我们描述了视网膜损伤的机械方法,该方法可确保对所有视网膜层的均匀损伤。除此之外,我们还描述了用于再生相关基因的体内操纵策略以及用于免疫组织化学分析的视网膜组织的制备。

【背景】视网膜是眼睛的感觉部分,任何物理或生理损伤都会导致视力下降。在进化过程中,高级脊椎动物失去了再生潜力,而原始脊椎动物具有巨大的能力来恢复失去的视力。研究诸如斑马鱼之类的原始生物中的再生事件可能是哺乳动物再生研究的希望。斑马鱼已用于视网膜再生研究,并使用了各种损伤范例。光漂白法会损伤感光细胞,而化学法会损伤神经节细胞层。刺伤法造成的机械损伤使所有视网膜层保持均匀的损伤。已经使用包括转基因方法在内的各种方法来寻找视网膜再生期间遗传事件的相关性。在这里,我们描述了用于mRNA转染的 in vivo 方法,该方法允许操纵高于内源性水平的基因表达水平。

关键字:斑马鱼, 视网膜, 电穿孔, 玻璃体内, 转染, 眼睛

材料和试剂

  1. 30G针(BD Microlance,30G½”,0.3 x 13毫米,目录号:304000)
  2. 离心管(Tarsons,目录号:500010x)
  3. 纸巾(Scott SCOTTFOLD毛巾,31.4厘米x 19.9厘米,货号:01960)
  4. 汉密尔顿注射器(Hamilton Company,10 µl,目录号:MICROLITER TM #701)
  5. 染色盒(通过将两个塑料移液器彼此平行固定在矩形塑料盒的底部进行定制)
  6. 玻璃载玻片(Superfrost Plus显微镜载玻片)(Fisher Scientific,目录号:12-550-15)
  7. pCS2 +质粒(大卫·特纳,密歇根大学,安阿伯分校)
  8. 3-氨基苯甲酸乙酯,甲烷磺酸盐,三卡因甲磺酸盐(Acros,目录号:118000500)
  9. 2x HBSS(从10x溶液中稀释,请参见食谱)
  10. mMESSAGE mMACHINE SP6套件(Thermo Fisher,目录号:AM1340)
  11. Lipofectamine messenger max试剂(Invitrogen,目录号:LMRNA001)
  12. DABCO(1,4二氮杂双环[2.2.2]辛烷,Sigma-Aldrich,目录号:D27802)
  13. Na 2 HPO 4 (磷酸氢二钠,Sigma-Aldrich,目录号:255793)
  14. NaH 2 PO 4 (磷酸二氢钠,Sigma-Aldrich,目录号:33198)
  15. KCl(氯化钾,Himedia,目录号:MB043)
  16. HEPES(Sigma-Aldrich,目录号:H3375)
  17. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  18. Tissue-Plus OCT复合物(Fisher Health care,目录号:4585)
  19. BSA(牛血清白蛋白组分-V,Himedia,目录号:GRM105)
  20. 海卫一X-100(西格玛奥德里奇,目录号:T8787)
  21. BrdU(5-Bromo-2'-deoxyuridine,Sigma-Aldrich,目录号:B5002)
  22. PFA(多聚甲醛,Sigma-Aldrich,目录号:P6148)
  23. PVA(聚乙烯醇,Sigma-Aldrich,目录号:P8136)
  24. 甘油(Sigma-Aldrich,目录号:G7757)
  25. Tris Base(Trizma基座,Sigma-Aldrich,目录号:T1503)
  26. HCl(盐酸,Sigma-Aldrich,目录号:320331)
  27. 蔗糖(Sigma-Aldrich,目录号:S0389)
  28. Tris-HCl(1 M,pH 7.5,100毫升)(请参阅食谱)
  29. 曲卡因甲磺酸盐溶液(100毫升)(请参阅食谱)
  30. 磷酸盐缓冲液(PB,1 M,pH 7.4,100毫升)(参见配方)
  31. 磷酸盐缓冲盐水(PBS,10x,pH 7.4,1 L)(请参阅食谱)
  32. HBSS(汉克斯平衡盐溶液,10倍,pH 7.14,100毫升)(请参阅食谱)
  33. 4%PFA(50毫升)(请参阅食谱)
  34. DABCO(2.5%,50毫升)(请参阅食谱)
  35. 蔗糖溶液(50毫升)(请参阅食谱)

设备

  1. 镊子(McPherson缝合带直捆扎平台钳10cm(4“)5 mm尺寸,钳口长度,Surtex仪器,目录号:FR-780-10)
  2. 立体显微镜(带有LED照明的Carl Zeiss TM Stemi TM DV4系列立体显微镜)
  3. 电穿孔器(Electro Square Porator,BTX哈佛仪器,型号:ECM 830)
  4. 电极(白金镊子,5 mm直径,45-0204电缆,BTX,目录号:45-0489)
  5. 低温恒温器(Leica,型号:CM3050 S)
  6. Rotospin旋转混合机(塔森)
  7. 摇水浴(Stuart,型号:SBS40)
  8. 离心机(渐新世,目录号:ScanSpeed 1736R)
  9. 尼康N i -E荧光显微镜和尼康A1共焦成像系统(Nikon A1-SHS,目录号:10225)

程序

  1. 视网膜损伤(Kaur等,2018; Mitra等,2018; Mitra等,2019; Sharma等等,2019)
    1. 在鱼水中准备50毫升1x三卡因甲磺酸盐溶液。
    2. 通过放入三卡因溶液来麻醉鱼,直到the的运动变慢。
    3. 将麻醉的鱼放在湿纸巾床上,右侧朝上。
    4. 将斑马鱼的眼睛聚焦在立体显微镜下,并用镊子轻轻倾斜眼球的背侧。
    5. 用30 G针刺伤伤口,刺伤眼睛的腹侧,然后损伤眼睛的另一侧。
    6. 通过从腹侧轻轻倾斜眼睛的背面来重复受伤,总共造成4次伤害。
    7. 让鱼在系统水中恢复活力(有关详细信息,请参见视频1)。


      视频1.斑马鱼的视网膜损伤

  2. 体内 mRNA转染(Mitra等,2018; Sharma等,2019)
    基因的体内过量表达是通过视网膜中的mRNA转染实现的。它涉及以下步骤(有关详细信息,请参见视频2):


    视频2.玻璃体内注射和吗啉代电穿孔

    1. 转染试剂的制备
      1. 将GFP或目标基因克隆到pCS2 +质粒中。从3'末端线性化包含插入片段的向量。使用sp6消息机器体外转录试剂盒制备mRNA。沉淀mRNA并溶解在无核酸酶的水中,以使库存量为2,000 ng / µl。
      2. 如下准备转染混合物(8 µl)和lipofectamine 2,000转染试剂:
        1. 在室温下将2μlmRNA(2,000 ng /μl)和2x HBSS溶液混合。
        2. 将2 µl lipofectamine与等体积的2x HBSS溶液按1:1的比例混合,然后将溶液保持在室温下。
        3. 让两种溶液在室温下静置五分钟。
        4. 以1:1的比例逐滴混合溶液(i)和(ii),并将混合物在室温下放置30分钟。使用包含500 ng / µl mRNA的最终转染混合物进行玻璃体内注射。
      注意:该转染混合物用于在4-5只斑马鱼中进行玻璃体内注射,并且仅使用了单个浓度的mRNA。根据实验需要,可以按比例放大或缩小这些体积,而不会影响转染效率。
    2. 玻璃体内注射体内过表达
      1. 用30 G的针头刺伤四个鱼,麻醉鱼并伤害右眼。
      2. 从第四次戳中,使用Hamilton注射器注入1 µl转染混合物。
      3. 将负电极放在右眼上,将正电极放在另一侧上,对鱼进行电穿孔。
      4. 在70 V电压下以五个脉冲电穿孔,持续时间为50毫秒,脉冲之间的间隔时间为950毫秒。
      5. 如E和F节所述,在受伤4天后收眼。
      6. 在基因过表达的视网膜中,与GFP转染的视网膜相比,检查BrdU阳性细胞的增殖情况(请参阅步骤E,F和G)。
      7. 通过对GFP进行免疫染色检查转染,随后进行成像,显示整个视网膜中的GFP。在此处附加了两个代表性图像,分别显示了在损伤部位和远离损伤部位的GFP表达(图1)。


        图1。显示GFP mRNA转染后斑马鱼视网膜中GFP的表达。星号标记了损伤点。ONL - 外核层; OPL - 外丛状层; INL - 内核层; IPL - 内丛状层; GCL - 神经节细胞层。

  3. 药物输送和吗啉代电穿孔(Kaur等,2018; Mitra等,2018; Mitra等,2019; Sharma et al。,2019)
    为了研究药物,蛋白质和基因敲低方法的局部作用,进行了玻璃体内注射。
    1. 注射液的制备:
      1. 将冻干的吗啉代(300 ng)溶于300 µl高压灭菌的Milli-Q水中,制成1 mM的储备液。使用纯净的1 mM溶液或用Milli-Q水稀释以使注射储备液为0.5或0.25 mM。
      2. 将蛋白质溶解在推荐的溶剂中制成储备液,然后在1x PBS或推荐的稀释剂中进一步稀释。
      3. 将药物溶于1 ml DMSO(二甲基亚砜)或推荐的溶剂中,以制成最终浓度的储备溶液,具体取决于药理抑制剂的分子量和重量。用Milli-Q水稀释,从主要原料中获得所需的工作浓度。
    2. 注射溶液:
      1. 如上所述,由于刺伤而伤及一个视网膜。
      2. 从第四次戳中,用Hamilton注射器注入约1 µl试剂。
      3. 对于吗啉代递送,电穿孔吗啉代以使其进入视网膜。
      4. 将正极放在注射吗啉代的眼睛上,将负极放在另一只眼睛上。
      5. 在上述条件下进行电穿孔。
      6. 如下所述,在视网膜损伤(dpi)后4天收获眼睛。

  4. BrdU标签
    BrdU脉冲标记完成3 h,然后以4 dpi采集眼睛。它通过在复制的DNA中掺入胸苷类似物(BrdU)来标记活跃增殖的细胞。
    1. 通过将15.35 mg BrdU粉末溶于10ml高压灭菌的Milli-Q水中,制成5 mM BrdU溶液。取1毫升等分试样,并保存在-20°C下供以后使用。
    2. 在使用前将BrdU溶液带入室温,然后填充胰岛素注射器。
    3. 麻醉鱼,并在显微镜下握住湿纸巾。
    4. 将胰岛素针轻轻插入骨盆鳍之间的中间,并使针与鱼体平行,注意不要损坏内部器官。
    5. 注入约一单位(15-20 µl)的BrdU溶液,并将鱼留在系统水中3 h。

  5. 收获和组织准备
    1. 收眼(有关详细信息,请参见视频3)
      1. 通过过量服用三卡因使鱼安乐死(长时间浸泡在1x三卡因溶液中)。
      2. 用镊子轻轻地将眼睛从插槽中拉出。
      3. 将眼睛放在培养皿中的冷冻固定液中,并在立体显微镜下聚焦。
      4. 用镊子握住眼睛,并用针刺穿角膜。在镊子的帮助下,撕开角膜并轻轻推动眼睛,使晶状体脱出。


      视频3.斑马鱼安乐死和眼睛收获

    2. 组织固定
      1. 在4%PFA中于4°C固定眼睛过夜,并在转子旋转时以12 rpm的速度连续旋转。
      2. 除去PFA,并在室温下连续旋转,以蔗糖梯度使组织脱水45分钟。添加蔗糖溶液,如下所示:
        5%蔗糖,1容量
        5%:20%蔗糖,2:1体积
        5%:20%蔗糖,1:1体积
        5%:20%蔗糖,1:2体积
        20%蔗糖,1容量
      3. 最后,用500 µl新鲜蔗糖溶液代替20%蔗糖,并向其中加入等体积的OCT。通过在室温下旋转混合30分钟。
      4. 通过包裹在肘上制成铝箔的立方体模具。
      5. 用OCT将模具填充至一半,在顶部放置标记的标记,并使眼睛与腹腔平行。
      6. 保持在-80°C,立即冻结眼睛。

  6. 冷冻切片和免疫染色
    1. 冷冻切片
      1. 打开低温恒温器,并在室内将视线障碍物保持10分钟。
      2. 借助OCT将组织固定在样品盘上。
      3. 将样品盘固定在样品头上并贴上载玻片标签。
      4. 去除多余的OCT,直到视网膜组织开始出现。
      5. 在载玻片上截取8-10 µm的薄串行部分。
      6. 让载玻片在室温下干燥过夜。
    2. 免疫染色
      1. 取一组幻灯片,将其水平放置在免疫染色架上。
      2. 用1x PBS洗涤3次,每次10分钟以去除OCT。
      3. 在室温下用PBST中的5%BSA(1x PBS和0.01%Triton X-100)封闭组织2-3小时。
      4. 除去封闭溶液,并在4°C下用一抗覆盖玻片过夜。
      5. 第二天收集一抗,并在室温下用PBST洗涤玻片3次,每次10分钟。
      6. 在室温下用荧光标记的二抗覆盖玻片2-3小时。
      7. 收集二抗并用PBST洗涤玻片三次。
      8. 用高压灭菌水停止反应。
      9. 让幻灯片在黑暗中垂直干燥10-15分钟。
      10. 用60 µl DABCO固定玻片并盖玻片。
      11. 让载玻片在暗室中于室温下干燥过夜。

  7. 图像采集
    在荧光显微镜下检查载玻片,并用共焦成像系统和配备荧光光学镜的尼康N i -E荧光显微镜和尼康A1共焦成像系统拍摄图像。

数据分析

通过观察视网膜切片中的荧光,在荧光显微镜下计数BrdU标记的细胞。在实验组和治疗组中,都对主要损伤部位的细胞进行计数,并避免这些斑点远离主要损伤。损伤点可以通过不连续的外核层来划分,如下所示(图2)。在每个实验设置中,至少要取三只眼。计数存在于三个视网膜部分的所有主要损伤部位中的标记细胞。排除异常值(极低和极高的值),并通过分析Excel工作表中的数据来计算平均值和标准偏差。


图2.显示带有红色标记的BrdU阳性细胞的损伤点(用星号标出)。损伤点周围的正方形标记了细胞计数区域。比例尺- 10 μ 米; ONL - 外核层; INL - 内核层; GCL - 神经节细胞层。

菜谱

  1. Tris-HCl(1 M,pH 7.5,100毫升)
    1. 将12.1 g Tris碱溶于80 ml Milli-Q水中,制成1 M Tris-HCl储备液
    2. 用浓盐酸溶液将pH调节至7.5,并用Milli-Q水将最终体积调节至100 ml
  2. 曲卡因甲烷磺酸盐溶液(100毫升)
    1. 量取2 g 3-氨基苯甲酸乙酯,甲磺酸盐并将其溶解在100 ml 0.1 M Tris-HCl(pH 7.5)溶液中(将10 ml 1 M溶液溶于90 ml碳酸氢钠中,将原液稀释至0.1 M)。 Milli-Q水)
    2. 使用前将溶液保持在4°C并在鱼类系统水中稀释(1:100)
  3. 磷酸盐缓冲液(PB,1 M,pH 7.4,100毫升)
    1. 称量以下成分:
      Na 2 HPO 4
      10.9877克
      NaH 2 PO 4
      2.711548 g
    2. 首先,将Na 2 HPO 4 溶解在80 ml Milli-Q H 2 O中,然后添加NaH 2 PO 4
    3. 用Milli-Q H 2 O调节pH到7.4并补足100 ml
    4. 高压灭菌溶液并用高压灭菌的Milli-Q水稀释至0.1 M的工作量
  4. 磷酸盐缓冲盐水(PBS,10x,pH 7.4,1 L)
    1. 测量以下组件:
      氯化钠
      75.97克
      Na 2 HPO 4
      9.937克
      NaH 2 PO 4
      3.59克
    2. 通过剧烈摇动或用磁珠将所有成分溶于800毫升Milli-Q水中
    3. 所有组分溶解后,将pH值调节至7.4,并补足最终体积至1,000 ml
    4. 对该溶液进行高压灭菌,并将其用作用Milli-Q水稀释的1x溶液
  5. HBSS(汉克斯平衡盐溶液,10倍,pH 7.14,100毫升)
    氯化钠
    8克
    氯化钾
    0.354克
    葡萄糖
    1.351克
    HEPES
    5.466克
    Na 2 HPO 4
    99.35毫克
    1. 测量这些成分并溶于80毫升Milli-Q水中
    2. 调节pH至7.14并补足最终体积至100 ml
    3. 用10倍的储备液制备2倍的溶液,并以1毫升等分试样在-20°C下储存
  6. 4%PFA(50毫升)
    1. 在50 ml干燥离心管中计量2 g PFA,然后向其中加入5 ml 1 M PB
    2. 用高压灭菌的Milli-Q水将体积调至45 ml,并在摇动的水浴中于65°C加热以溶解PFA
    3. 中间取下离心管,剧烈颠倒混合沉降的粉末。完全溶解大约需要15-30分钟
    4. 适当溶解后,用高压灭菌的Milli-Q水将体积调节至50 ml,并立即置于冰上。要长期保存,请分装少量样品并保存在-20°C下
  7. DABCO(2.5%,50毫升)
    1. 在50 ml离心管中量取12 g甘油
    2. 量取4.8克PVA,并将其添加到甘油中。轻轻颠倒试管至均匀溶液,充分混合
    3. 加入12 ml高压灭菌的Milli-Q水,并在转子上混合过夜。然后在室温下旋转
    4. 加入24 ml 0.2 M Tris-HCl,pH 8-8.5
      1. 将12.1 g Tris碱溶于80 ml Milli-Q水中,制成100 ml 1 M Tris-HCl溶液
      2. 用浓盐酸溶液将pH值调节至8.2,并补足最终体积至100 ml
      3. 要制成0.2 M Tris-HCl,请在24 ml Milli-Q水中取6 ml 1 M储备液
    5. 将溶液在摇床水浴中加热至50°C,持续15-30分钟,然后以5,000 x g 离心15分钟
    6. 小心地除去上清液,并向上清液中加入1.25 g DABCO
    7. 以5,000 x g 离心15分钟
    8. 将上清液分装在1 ml试管中,并将其储存在-20°C下以备后用
  8. 蔗糖溶液(50毫升)
    1. 5%蔗糖:量取2.5克蔗糖并将其溶解在40毫升0.1 M PB溶液中。用0.1 M PB将体积调节至50 ml
    2. 20%蔗糖:量取10克蔗糖并将其溶解在40毫升0.1 M PB溶液中。旋转溶解后,用0.1 M PB将体积调节至50 ml
    3. 将溶液储存在4°C以便长期使用

致谢

PS感谢Wellcome Trust / DBT印度联盟和IISER Mohali提供的博士后研究金。PS感谢本科生Ajay Kumar进行视频捕获和编辑。这项工作得到了Rajesh Ramachandran授予的Wellcome Trust / DBT印度联盟中级研究金(IA / I / 12/2/500630)的支持。RR还感谢印度DST(EMR / 2017/001816),DBT印度(BT / PR9407 / BRB / 10/12612013),(BT / PR17912 / MED / 31/336)科学教育和研究委员会(SERB)的研究资金/ 2016)和IISER Mohali的支持。

利益争夺

作者宣称他们没有利益冲突。

参考文献

  1. Kaur,S.,Gupta,S.,Chaudhary,M.,Khursheed,MA,Mitra,S.,Kurup,AJ和Ramachandran,R.(2018)。 let-7 microRNA介导的shh信号调节和基因调节网络对于视网膜再生至关重要。 Cell Rep 23(5):1409-1423。
  2. Mitra,S.,Sharma,P.,Kaur,S.,Khursheed,MA,Gupta,S.,Ahuja,R.,Kurup,AJ,Chaudhary,M.,and Ramachandran,R.(2018)。 Histone脱皮酶重新编程的希拉里娜(Merler glia)inlin重新编程介导的Minler glylia iScience 7:68-84。
  3. Mitra,S.,Sharma,P.,Kaur,S.,Khursheed,MA,Gupta,S.,Chaudhary,M.,Kurup,AJ和Ramachandran,R.(2019)。在斑马鱼视网膜再生过程中,Myc必须对lin28a进行双重调节。 J Cell Biol 218:489-507。
  4. Sharma,P.,Gupta,S.,Chaudhary,M.,Mitra,S.,Chawla,B.,Khursheed,MA和Ramachandran,R.(2019)。 Oct4介导斑马鱼视网膜再生过程中的Muller胶质细胞重编程和细胞周期退出。 生命科学联盟 2(5):pii:e201900548。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Sharma, P. and Ramachandran, R. (2019). Retina Injury and Retina Tissue Preparation to Study Regeneration in Zebrafish. Bio-protocol 9(24): e3466. DOI: 10.21769/BioProtoc.3466.
  2. Mitra, S., Sharma, P., Kaur, S., Khursheed, M.A., Gupta, S., Chaudhary, M., Kurup, A.J., and Ramachandran, R. (2019). Dual regulation of lin28a by Myc is necessary during zebrafish retina regeneration. J Cell Biol 218: 489-507.
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