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A Streamlined Method for the Preparation of Gelatin Embedded Brains and Simplified Organization of Sections for Serial Reconstructions
脑组织明胶包埋的最新方法和用以连续重建的切片组织的简化排列   

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Abstract

Gelatin embedding of whole brains for sectioning is a critical procedure used in neuroscience to ensure all morphological and spatial details are preserved intact. Here, we describe an inexpensive, reproducible and efficient means to embed post-fixed brains ready for sectioning in gelatin within a week’s time. The sections obtained are distortion-free and their fragile internal structures preserved which can be used for serial reconstructions for lesion studies and mapping of viral expression after stereotaxic injections. In addition, the separation of adjacent slices into a series of 3-4 vials facilitates subsequent organization and assembly of serial sections at the mounting step.

Keywords: Gelatin embedding(明胶包埋), Serial reconstruction(连续重建), Slide mounting(封片), Lesion(病变), Viral expression(病毒表达), Neuronal tracing(神经元示踪)

Background

Recent advances in behavioral neurosciences have allowed the introduction of opsins and antibody targeted-toxins to specific subsets of neurons and regions of the brain. These studies often require visualization of whole brain sections for histological and morphological analysis to localize cell-type specific antibody targeted-toxin induced lesions by stereotaxic injection for behavioral validation (Aoki et al., 2015) or the introduction of virus delivered transgenes (Aquili et al., 2014). Detailed mapping of neuronal circuitry by rabies virus (Suzuki et al., 2012) and localization surveys of serial sections have been achieved using gelatin as an embedding agent, which acts a structural substrate within and around the tissue. Gelatin impregnated brain tissue provides strengthened support for delicate internal structures such as the hippocampus and ventricle spaces which are easily damaged when processed for immunohistochemistry (IHC) and subsequent mounting onto slides. Embedded brain sections are also often free of distortions when mounted and adjacent sections can be used for serial reconstructions.

Qualities of the gel embedding and post-IHC handling are dependent on several procedural details, which can be tedious and time consuming. In previous published protocols, proper penetration and infiltration of the gelatin into the brain and ventricular spaces required a vacuum oven (Griffioen et al., 1992). Further, embedding the brains in a small mold is challenging as the brain has a tendency to float. Additionally, identifying and orienting adjacent slices after IHC processing for serial reconstructions can be arduous.

In this improved protocol, we address several issues important for timely and trouble-free gelatin embedding of whole brains. Begin with rapid impregnation of gelatin into the brain using a magnetic stir bar to weigh the brain down into the liquefied gelatin. Further, the use of an icebox allows the gelatin to set from bottom to top thus eliminating the problem of floating brains. Finally, the arrangement and determination of adjacent serial sections for 3D reconstruction series are simplified by placing the adjacent sections sequentially into 3 to 4 vials, looping back to the first vial after the last. After IHC processing, the sections from each vial are placed in a column next to one another in a large Petri-dish and adjacent slices can be rapidly mounted moving along row by row.

Materials and Reagents

  1. 50 ml conical tubes in styrofoam frack (Corning, Falcon®, catalog number: 352098 )
  2. Weigh boats, small and medium (Dyn-A-Med Plus, catalog numbers: 80051 , 80056 )
  3. 150 mm Petri dishes (Corning, Falcon®, catalog number: 351058 )
  4. PS-10 vials (AS ONE, catalog number: 9-892-12 )
  5. Frosted slides (Matsunami Glass, catalog number: S024410 )
  6. Single edge razors (FEATHER Safety Razor, catalog number: 99129 )
  7. Paintbrush (Arteje Brush Camlon Pro, model 630 #3/0 Round)
  8. Kimwipe
  9. Paraformaldehyde (Merck, catalog number: 104005 )
  10. Sodium phosphate dibasic (Na2HPO4) (Wako Pure Chemical Industries, catalog number: 197-09705 )
  11. Sodium phosphate monobasic (NaH2PO4) (Wako Pure Chemical Industries, catalog number: 197-02865 )
  12. Sucrose (Wako Pure Chemical Industries, catalog number: 190-00013 )
  13. Gelatin (Wako Pure Chemical Industries, catalog number: 077-03155 )
  14. H2O2 (Wako Pure Chemical Industries, catalog number: 081-04215 )
  15. Triton X-100 (Bio-Rad Laboratories, catalog number: 1610407 )
  16. Tween-20 (Bio-Rad Laboratories, catalog number: 1706531 )
  17. Anti-tyrosine hydroxylase (Enzo Life Sciences, catalog number: BML-SA497-0100 )
  18. Thionin (Alfa Aesar, catalog number: A18912 )
  19. Standard ABC Peroxidase Kit (Vector Laboratories, catalog number: PK-4000 )
  20. Metal Enhanced DAB Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 34065 )
  21. Entellan new mounting medium (Merck, catalog number: 107961 )
  22. Rabbit Anti-ChAT conjugated saporin (Advanced Targeting Systems, catalog number: IT-42 )
  23. Mouse Anti-NeuN (Abcam, catalog number: ab104224 )
  24. Goat Anti-ChAT (EMD Millipore, catalog number: AB144P )
  25. Biotin conjugated Goat anti-rabbit IgG (Thermo Fisher Scientific, catalog number: B-2770 )
  26. Biotin conjugated Goat anti-mouse IgG (Thermo Fisher Scientific, catalog number: B-2763 )
  27. Biotin conjugated Rabbit anti-goat IgG (Thermo Fisher Scientific, catalog number: A10518 )
  28. 4% PFA/0.1 M PB (4% paraformaldehyde/0.1 M phosphate buffer, pH 7.4) (see Recipes)
  29. 0.1 M PB (0.1 M phosphate buffer, pH 7.4) (see Recipes)
  30. 30% sucrose 0.1 M phosphate buffer, pH 7.4 (see Recipes)
  31. 4% paraformaldehyde/10% sucrose in 0.1 M phosphate buffer, pH 7.4 (see Recipes)
  32. 0.05 M PB (0.05 M phosphate buffer, pH 7.4) (see Recipes)
  33. 30% sucrose 0.05 M phosphate buffer, pH 7.4 (see Recipes)
  34. Blocking solution (see Recipes)
  35. Antibody solution (see Recipes)
  36. 0.5% w/v gelatin in H2O (see Recipes)

Equipment

  1. Rack for 50 ml conical tubes
  2. Thermometer
  3. Spoon spatula
  4. Spin-plus 19 mm magnetic stir-bars (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F37144-0034 ) and magnetic spin-bar removal tool
  5. 2 x 500 ml beakers (DWK Life Sciences, Duran®, catalog number: 21 106 48 )
  6. 5 L liquid waste buckets for PFA/gel (AS ONE, catalog number: 4-5308-03 )
  7. Bent forceps (Ideal-Tek, model: 650.S.6 )
  8. Oven (SANYO, model number: MIR-162 )
  9. Hot plate with magnetic stirring capability (IKA, model: C-MAG HS 7 )
  10. Refrigerator (SANYO, model: MPR-414FR )
  11. Fume hood
  12. Vibratome (Leica Biosystems, model: Leica VT1000 S )
  13. Upright light microscope (Olympus, model: CX22LED )

Procedure

  1. Gelatin embedding
    1. Wash 4% PFA/0.1 M PB (see Recipes), perfused, 72 h post-fixed brains, once with 0.1 M phosphate buffer (PB) (see Recipes) and immerse brain in 30% sucrose/0.1 M PB (see Recipes) in a 50 ml conical tube. Use magnetic stir-bar atop a 15 ml inverted conical tube cap to help keep brain submerged in the tube. Incubate at 4 °C overnight (18 h).
    2. Decant 30% sucrose/0.1 M PB and replace with 50 °C double distilled water (ddH2O) and put tubes containing brains in a 55 °C incubator for 45 min to equilibrate the temperature of the brains. Continue to use magnetic stir-bar and conical cap to submerge brain for all subsequent steps between 50-55 °C.
    3. While brains are being temperature equilibrated, pre-warm hot plate for gelatin preparation, make sure temperature of the hot plate does not exceed 60 °C.
    4. Make 14% gelatin/10% sucrose solution (25 ml/brain); for 500 ml (20 brains), add 70 g gelatin little by little to 450 ml of 50 °C ddH2O using magnetic stir bar speed from slow to fast, which will pull in gelatin without causing clumps (Video 1).
    5. Once the gelatin is dissolved, add 50 g of sucrose and bring volume to 500 ml with 50 °C ddH2O.
    6. Make 7% gelatin/5% sucrose solution by pre-warming 150 ml of ddH2O to 55 °C and add 150 ml of 14% gelatin/10% sucrose to the pre-warmed ddH2O, keep all gelatin solutions at 55 °C in oven (Video 1).

      Video 1. Gelatin solution preparation. Visual demonstration of 14% gelatin/10% sucrose and 7% gelatin/5% sucrose preparation is shown in this example.

    7. Decant warm ddH2O submerging the brains in 7% gelatin solution/5% sucrose, swirl, incubate at 55 °C for 2 h (Video 2).

      Video 2. Brain submersion in gelatin. Visual demonstration of removal of solutions and submersion in gelatin using magnetic stir bar is shown in this example.

    8. Decant 7% gelatin solution/5% sucrose submerging the brains in 14% gelatin solution/10% sucrose, swirl, and incubate at 55 °C for 3 h.
    9. Mark weigh boats with animal ID numbers.
    10. Using a spoon spatula, work swiftly and carefully dislodge brain and liquid contents into a weigh boat atop a large Petri dish on a bed of ice. Top off with gelatin, remove bubbles, orient the brain and gently press down with bent forceps to keep it submerged until the bottom begins to set (about 7-10 min, Video 3).

      Video 3. Casting of gelatin embedded brain and removal after gelatin setting. Visual demonstration of casting gelatin embedded brain in weigh boat mold and subsequent cutting of set mold from weigh boat are shown in this example.

    11. Move brains to a 4 °C refrigerator and leave for another 90 min.
    12. Mark sample vials with animal ID numbers.
    13. Carefully cut out the casted brain with a new single edge razor blade and slide into sample vial.
    14. Add 4% PFA/10% sucrose in 0.1 M PB (see Recipes) to the vial with brain, place the vial in a refrigerator overnight (18 h).
    15. Remove PFA, rinse once with 0.05 M PB (see Recipes) and replace with 30% sucrose/0.05 M PB (see Recipes). Keep sample vials in the refrigerator for a minimum of 72 h.

  2. Sequential slice collection
    1. After embedding, mark 3 to 4 vials for each animal. (i.e., animal ID–#1 to 3 or 4)
    2. Cut block that includes the region of interest, trim block and systematically cut diagonal corners to orient the slices correctly during mounting (i.e., upper right and lower left corners relative to the face of the block).
    3. Collect sections from microtome or vibratome in 0.05 M PB and place sections sequentially among the 3 to 4 vials. Loop back to the first vial after the third or fourth vial (Figure 1). Sections between 40-80 μm from the vibratome generally work the best.


      Figure 1. Slice collection schematic from sectioning device and post-immunostaining arrangement of slices at the mounting step. Slices are collected sequentially in numbered vials as shown in the figure. After the completion of immunostaining procedure, each of the vial’s slices is arranged in columns in a large Petri dish, with the next vial’s column arranged beside the previous vial. Once arranged, serial mounting is achieved by moving along row by row. Arrangement by sequential vial collection is simpler since the distance between slices is greater. This allows for slices to be easily distinguished from one another more accurately and thus, mounting is performed more rapidly.

  3. Immunostaining
    Users’ choice: fluorescent labeling or IHC. Briefly, the examples shown in Figures 2 and 3 were stained by the following procedures.
    1. Pretreat the samples with 3% H2O2 in 0.05 M PB for 20 min, and then incubate the samples with blocking solution (see Recipes) for 1 h.
    2. Decant the blocking solution and add primaries in antibody solution (see Recipes, 1:100 [anti-ChAT]; 1:1,000 [anti-TH], 1:1,000 [anti-NeuN]), incubate at 4 °C for 48 h.
    3. Decant the primaries. Wash with PBS for 5 min, 3 times.
    4. Add biotin-conjugated secondary antibodies in antibody solution (1:400 anti-goat, ChAT, anti-rabbit for TH and anti-mouse for NeuN), incubate at 25 °C for 4 h.
    5. Decant the secondaries. Wash with PBS for 5 min, 3 times.
    6. Add Standard ABC Peroxidase solution diluted according to manufacturer’s protocol in PBS and 0.2% Tween-20, incubate at 25 °C for 4 h.
    7. Decant the ABC Peroxidase solution. Wash with PBS for 5 min, 3 times.
    8. Make DAB working solution according to the manufacturer’s protocol and add to slices. Wait approximately 3-5 min for signal development and stop reaction with four 0.1 M PB washes.
    9. Decant ABC peroxidase solution and wash with PBS for 5 min, 3 times.
    10. Store slices in 0.05 M PB.

  4. Serial mounting after immunostaining
    1. Dump contents of first vial into large Petri dish with 0.01 M PB (1:5 dilution of 0.05 M PB in H2O) and arrange in a column using a fine paintbrush.
    2. Carefully move contents of second vial and arrange in column next to first vial’s column.
    3. Repeat until all vials are arranged in columns next to one another.
    4. Mount adjacent serial slices on slides with a paintbrush, moving along row by row (Figures 1 and 2).
    5. If dry mounting IHC slices, prepare slide by coating with 0.12% w/v gelatin in ddH2O (dilute 1:4 0.5% w/v gelatin in ddH2O, see Recipes) with a paintbrush in a medium sized weigh boat. Leave slide half submerged by resting the frosted side on the square edge of the weigh boat and the submerged side on the bottom slope of the spout (Video 4).
    6. Move initial slices from large Petri dish to submerged slide in medium-sized weigh boat containing 0.12% gelatin by placing slice in the solution, orienting the slice and then carefully sliding the slice up to its proper position away from the submerged edge of the slide.
    7. Push the slide towards the spout of the weigh boat, exposing more of the slide from the 0.12% gelatin and repeating the process in step D6 for the adjacent slices to fill the rest of the slide.
    8. Blot dry edge of slide on Kimwipe and air dry for 24 h at 22 °C before counterstaining, dehydration, mounting medium and cover slip placement.

      Video 4. Mounting of sections on a slide. Visual demonstration of mounting gelatin embedded brain slices in a weigh boat filled with 0.12% w/v gelatin. In this example, only one of four vials was used for mapping antibody-mediated toxin deletions (each slice is 240 μm apart) and sequentially arranged from top left, moving along top to bottom, left to right.


      Figure 2. Cannula placement localized in the ventral tegmental area (VTA) using serial sections. Gelatin embedded rat brain slices were stained with anti-tyrosine hydroxylase visualized with horseradish peroxidase and 3’,3’-diaminobenzidine (DAB). Slices were dry mounted onto a slide, counterstained with 0.02% thionin for 3 minutes, run through an ethanol dehydration series and cover-slipped with Entellan mounting medium. Six 80 μm serial slices were mounted sequentially starting from top left moving along horizontally ending on the bottom right and the vial and slice number are labeled above the slice (see Figure 1 for origin of vial and slice).

Data analysis

Localization of cannula placement in the rat VTA was verified using an Olympus CX22LED light microscope (Figure 2). Additionally, verification and mapping of the extent of stereotaxically injected anti-ChAT-saporin antibody directed lesions in the rat striatum (Aoki et al., 2015, Figure 3) were analyzed qualitatively by using an Olympus CX22LED light microscope and anatomical landmarks from a rat brain atlas (Paxinos and Watson, 2004).


Figure 3. Verification and lesion mapping of antibody targeted immunotoxin delivery to cholinergic neurons in the rat striatum. A. Representative coronal sections of rat striatal DAB stained slices display mostly intact NeuN staining but cell-specific deletion of cholinergic interneurons in ChAT staining among rabbit anti-ChAT-saporin mediated lesioned cases (arrows–dorsal medial stratum, DMS or ventral striatum, VS). Scale bars = 1 mm. B. An example of a limited nonspecific lesion indicated by a dashed area (NeuN) where fewer cells appear and an example more extensive non-specific deletion using a non-selective goat IgG-saporin injection as a control (arrow). C and D. The most restricted (solid black) and the broadest areas (gray) of cholinergic interneuronal deletions in each of the behavioral conditions are shown. Coordinate distances of the slices from the bregma are indicated to the left. The extent of the lesions in DMS (C) and VS (D) appears similarly between all conditions. Anatomical landmarks in the DAB stained photographs: LV, lateral ventricle; CC, corpus callosum; AC, anterior commissure. Figure modified for clarification and reprinted with permission (Aoki et al., 2015).

Notes

It is strongly suggested that vials and weigh boats are marked before hand with animal IDs and that the transfer of the brain from one container to another is done carefully as to ensure there is no confusion of the identity of the individual.

Recipes

  1. 4% PFA/0.1 M PB (4% paraformaldehyde/0.1 M phosphate buffer, pH 7.4)
    4% w/v paraformaldehyde in ddH2O
    80 mM Na2HPO4
    20 mM NaH2PO4
  2. 0.1 M PB (0.1 M phosphate buffer, pH 7.4)
    80 mM Na2HPO4 in ddH2O
    20 mM NaH2PO4
  3. 30% sucrose/0.1 M PB (30% sucrose 0.1 M phosphate buffer, pH 7.4)
    30% w/v sucrose in ddH2O
    80 mM Na2HPO4
    20 mM NaH2PO4
  4. 4% paraformaldehyde/10% sucrose in 0.1 M phosphate buffer, pH 7.4
    4% w/v paraformaldehyde in ddH2O
    20% w/v sucrose
    80 mM Na2HPO4
    20 mM NaH2PO4
  5. 0.05 M PB (0.05 M phosphate buffer, pH 7.4)
    40 mM Na2HPO4 in ddH2O
    10 mM NaH2PO4
  6. 30% sucrose 0.05 M phosphate buffer, pH 7.4
    30% w/v sucrose in ddH2O
    40 mM Na2HPO4
    10 mM NaH2PO4
  7. Blocking solution
    1x PBS
    5% secondary’s host serum
    0.2% Triton X-100
  8. Antibody solution
    1x PBS
    2% secondary’s host serum
    0.2% Triton X-100
  9. 0.5% w/v gelatin in ddH2O
    ddH2O to 50 °C
    0.5% w/v gelatin

Acknowledgments

We thank Dr. Tom Ruigrok who provided the initial protocols for gelatin embedding and Mayank Aggarwal for allowing us to display the stained slide of cannula placement in the VTA used in Figure 2. We also greatly appreciate François Beauchain for critical proofreading and Yumiko Akamine for assistance with the video production. Finally, we tip our hats to Andres Carrasco for calling our attention Bio-protocol.
Support from the Human Frontier Science Program (A.W.L., J.R.W.), JSPS Grant-in-Aid for Challenging Exploratory Research, Grant-in-Aid for JSPS Fellows and Grant-in-Aid for Young Scientists–category A (S.A.) made research performed in this publication possible. The authors declare no competing financial interests.

References

  1. Aoki, S., Liu, A. W., Zucca, A., Zucca, S. and Wickens, J. R. (2015). Role of striatal cholinergic interneurons in set-shifting in the rat. J Neurosci 35(25): 9424-9431.
  2. Aquili, L., Liu, A. W., Shindou, M., Shindou, T. and Wickens, J. R. (2014). Behavioral flexibility is increased by optogenetic inhibition of neurons in the nucleus accumbens shell during specific time segments. Learn Mem 21(4): 223-231.
  3. Griffioen, H. A., Van der Beek, E. and Boer, G. J. (1992). Gelatin embedding to preserve lesion-damaged hypothalami and intracerebroventricular grafts for vibratome slicing and immunocytochemistry. J Neurosci Methods 43(1): 43-47.
  4. Paxinos, G. and Watson, C. (2004). The rat brain in stereotaxic coordinates. Elsevier Academic Press.
  5. Suzuki, L., Coulon, P., Sabel-Goedknegt, E. H. and Ruigrok, T. J. (2012). Organization of cerebral projections to identified cerebellar zones in the posterior cerebellum of the rat. J Neurosci 32(32): 10854-10869.

简介

整个脑切片明胶嵌入是神经科学中使用的关键程序,以确保所有的形态和空间的细节保存完好。 在这里,我们描述了一个廉价,可重复和有效的方法来嵌入后固定的大脑准备切片明胶一个星期的时间。 获得的部分是无畸变的,它们的脆弱内部结构被保留下来,可用于系列重建病变研究和立体定位注射后的病毒表达图谱。 此外,将相邻切片分离成3-4个小瓶系列便于在安装步骤中对连续切片进行组织和组装。
【背景】行为神经科学的最新进展已经允许将视蛋白和抗体靶向毒素引入特定亚群的神经元和大脑区域。这些研究通常需要全脑切片的可视化用于组织学和形态学分析,以通过用于行为验证的立体定位注射(Aoki等,,2015)或引言定位细胞类型特异性抗体靶向毒素诱导的损伤的病毒递送转基因(Aquili等人,2014)。利用狂犬病病毒(Suzuki等人,2012)对神经元回路进行了详细的绘图,并且使用明胶作为包埋剂实现了连续切片的定位调查,所述包埋剂作为组织内和周围的结构基质。明胶浸渍的脑组织提供了加强的支持精致的内部结构,如海马和脑室空间,这是容易损坏免疫组织化学(IHC)处理和随后安装到幻灯片上。嵌入式脑切片在安装时通常也不会变形,并且可以将相邻切片用于连续重建。

凝胶嵌入和IHC处理后的质量取决于几个程序性的细节,这可能是乏味和耗时的。在先前公开的方案中,明胶向脑和心室空间的适当渗透和渗透需要真空烘箱(Griffioen等,1992)。此外,将大脑嵌入小模具中是具有挑战性的,因为大脑具有漂浮的趋势。另外,在连续重构的IHC处理之后识别和定向相邻切片可能是艰巨的。

在这个改进的协议中,我们解决了几个重要的问题,以便及时和无故障的明胶包埋整个大脑。首先用磁力搅拌棒将明胶快速浸渍到大脑中,将大脑称重到液化的明胶中。此外,使用冰箱可使明胶自下而上设置,从而消除浮动脑部的问题。最后,通过将相邻的部分依次放入3到4个小瓶中,并在最后一个之后环回到第一个小瓶,来简化3D重建序列的相邻连续部分的布置和确定。在IHC处理之后,将每个小瓶的切片放置在一个大的培养皿中彼此相邻的列中,并且相邻的切片可以快速安装成逐行移动。

关键字:明胶包埋, 连续重建, 封片, 病变, 病毒表达, 神经元示踪

材料和试剂

  1. 聚苯乙烯泡沫塑料瓶中的50ml锥形管(Corning,Falcon ,产品目录号:352098)
  2. 称量船,中小型(Dyn-A-Med Plus,目录号:80051,80056)
  3. 150毫米培养皿(Corning,Falcon ,产品目录号:351058)
  4. PS-10小瓶(AS ONE,目录号:9-892-12)
  5. 磨砂玻璃(松浪玻璃,产品目录号:S024410)
  6. 单刃剃须刀(FEATHER安全剃刀,目录号:99129)
  7. 画笔(Arteje刷Camlon临,模型630#3/0回合)
  8. Kimwipe
  9. 多聚甲醛(Merck,目录号:104005)
  10. 磷酸二氢钠(Na 2 HPO 4)(Wako Pure Chemical Industries,目录号:197-09705)
  11. 磷酸二氢钠(NaH 2 PO 4)(Wako Pure Chemical Industries,目录号:197-02865)
  12. 蔗糖(Wako Pure Chemical Industries,目录号:190-00013)
  13. 明胶(Wako Pure Chemical Industries,目录号:077-03155)
  14. (和光纯药工业,目录编号:081-04215)
  15. Triton X-100(Bio-Rad Laboratories,目录号:1610407)
  16. 吐温-20(Bio-Rad Laboratories,目录号:1706531)
  17. 抗酪氨酸羟化酶(Enzo Life Sciences,目录号:BML-SA497-0100)
  18. 硫翁(Alfa Aesar,目录号:A18912)
  19. 标准ABC过氧化物酶试剂盒(Vector Laboratories,目录号:PK-4000)
  20. 金属增强型DAB试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:34065)
  21. Entellan新安装介质(Merck,目录号:107961)
  22. 兔抗ChAT结合皂素(高级靶向系统,目录号:IT-42)
  23. Mouse Anti-NeuN(Abcam,产品目录号:ab104224)
  24. 山羊抗ChAT(EMD Millipore,目录号:AB144P)
  25. 生物素结合的山羊抗兔IgG(Thermo Fisher Scientific,目录号:B-2770)
  26. 生物素缀合的山羊抗小鼠IgG(Thermo Fisher Scientific,目录号:B-2763)
  27. 生物素缀合的兔抗山羊IgG(Thermo Fisher Scientific,目录号:A10518)
  28. 4%PFA / 0.1M PB(4%多聚甲醛/0.1M磷酸盐缓冲液,pH7.4)(见食谱)
  29. 0.1M PB(0.1M磷酸盐缓冲液,pH7.4)(见配方)
  30. 30%蔗糖0.1M磷酸盐缓冲液,pH7.4(见食谱)
  31. 4%多聚甲醛/ 10%蔗糖在0.1M磷酸盐缓冲液,pH7.4中(参见食谱)
  32. 0.05M PB(0.05M磷酸盐缓冲液,pH7.4)(见食谱)
  33. 30%蔗糖0.05M磷酸盐缓冲液,pH7.4(见食谱)
  34. 阻止解决方案(见食谱)
  35. 抗体溶液(见食谱)
  36. 在H 2 O中的0.5%w / v明胶(参见配方)

设备

  1. 支架50毫升锥形管
  2. 温度计
  3. 勺子刮刀
  4. Spin-plus 19毫米磁力搅拌棒(SP Scienceware-Bel-Art Products-H-B Instrument,目录号:F37144-0034)和磁性旋转棒去除工具
  5. 2×500毫升烧杯(DWK生命科学公司,Duran ,产品目录号:2110648)
  6. 用于PFA /凝胶(AS ONE,目录号:4-5308-03)的5L废液桶
  7. 弯钳(Ideal-Tek,型号:650.S.6)
  8. 烤箱(SANYO,型号:MIR-162)
  9. 具有磁力搅拌功能的热板(IKA,型号:C-MAG HS 7)
  10. 冰箱(SANYO,型号:MPR-414FR)
  11. 通风柜
  12. 颤音(Leica Biosystems,型号:Leica VT1000 S)
  13. 直立式光学显微镜(奥林巴斯,型号:CX22LED)

程序

  1. 明胶嵌入
    1. 用0.1M磷酸盐缓冲液(PB)(见食谱)洗涤4%PFA / 0.1M PB(参见食谱),灌注72小时后固定的大脑,并将脑浸入30%蔗糖/0.1M PB(参见食谱)在50ml锥形管中。使用磁力搅拌棒顶部15毫升倒锥形管帽,以帮助保持大脑淹没在管。
      4°C孵育过夜(18小时)。
    2. 倾倒30%蔗糖/0.1M PB,并用50℃双蒸水(ddH 2 O)代替,并将包含脑的管在55℃培养箱中培养45分钟以平衡大脑的温度。继续使用磁力搅拌棒和圆锥形帽来淹没大脑,在50-55°C之间的所有后续步骤。
    3. 当大脑温度达到平衡时,预热明胶制备热板,确保热板温度不超过60°C。
    4. 制成14%明胶/ 10%蔗糖溶液(25ml /脑);对于500ml(20个脑),使用磁力搅拌棒从缓慢到快速的速度将70g明胶逐渐添加到450ml的50℃ddH 2 O中,这将拉入明胶而不引起团块(视频1)。
    5. 一旦明胶溶解后,加入50克蔗糖,并用50℃ddH 2 O使体积达到500毫升。
    6. 通过将150ml ddH 2 O预热至55℃制备7%明胶/ 5%蔗糖溶液,并将150ml 14%明胶/ 10%蔗糖加入到预热的ddH > 2 O,将所有明胶溶液置于55°C烘箱(视频1)。

      视频1

    7. 滗析温热的ddH 2 O将大脑浸入7%明胶溶液/ 5%蔗糖中,旋转,在55℃孵育2小时(视频2)。

      视频2

    8. 倒入7%明胶溶液/ 5%蔗糖,将其浸入14%明胶溶液/ 10%蔗糖中,旋转,并在55℃下孵育3小时。
    9. 马克用动物身份证号码称重船只。
    10. 使用勺子刮刀,迅速并小心地将大脑和液体内容物移入冰块上的大培养皿顶上的称重船。取下泡沫,取出大脑,用弯钳轻轻压下,直到底部开始凝固(大约7-10分钟,视频3)。

      视频3

    11. 将大脑移动到4°C的冰箱,再等待90分钟。
    12. 用动物ID号标记样品瓶。

    13. 用新的单刃刀片小心切出铸造的大脑,并将其滑入样品瓶中。
    14. 将0.1M PB中的4%PFA / 10%蔗糖(见食谱)加入装有脑的小瓶中,将小瓶置于冰箱中过夜(18小时)。
    15. 去除PFA,用0.05M PB(参见食谱)冲洗一次,用30%蔗糖/0.05M PB代替(见食谱)。保持样品瓶在冰箱至少72小时。

  2. 顺序切片收集
    1. 包埋后,每只动物标记3至4个小瓶。 (即,动物ID-#1至3或4)
    2. 切割包括感兴趣区域的块,修剪块并系统地切割对角线,以便在安装期间正确定位切片( ie ,相对于块的正面的右上角和左下角)。 />
    3. 收集0.05 M PB切片机或vibratome切片,并连续放置3至4小瓶。在第三个或第四个小瓶后回到第一个小瓶(图1)。
      从vibratome的40-80微米之间的部分通常效果最好。


      图1.安装步骤中切片装置和切片后免疫染色装置的切片收集示意图切片按照图中所示顺序收集在编号的小瓶中。完成免疫染色程序后,将每个小瓶的切片放置在一个大培养皿中的柱中,下一个小瓶的柱布置在前一个小瓶的旁边。一旦安排,串行安装是通过逐行移动来实现的。由于切片之间的距离更大,顺序瓶收集的布置更简单。这样就可以更加准确地将切片彼此区分开来,从而更快速地进行安装。

  3. 免疫染色
    用户选择:荧光标记或IHC。简而言之,图2和3中所示的实例通过以下程序染色。
    1. 用0.05%PB预处理样品3%H 2 O 2 20分钟,然后将样品与封闭溶液一起孵育1小时(参见食谱)。 />
    2. 滗析封闭溶液并在抗体溶液中添加原色(参见配方,1:100 [抗ChAT]; 1:1,000 [抗-TH],1:1,000 [抗NeuN]),在4℃孵育48小时。
    3. 颂扬初选。用PBS清洗5分钟,3次。
    4. 在抗体溶液(1:400抗山羊,ChAT,抗TH的抗兔和NeuN的抗小鼠)中加入生物素结合的二抗,在25°C孵育4 h。
    5. 斟酌二手。用PBS清洗5分钟,3次。
    6. 加入标准的ABC过氧化物酶溶液,按照制造商的方案稀释在PBS和0.2%Tween-20中,在25℃孵育4小时。
    7. 滗ABC ABC过氧化物酶溶液。用PBS清洗5分钟,3次。
    8. 根据制造商的协议制作DAB工作解决方案并添加到切片中。等待约3-5分钟的信号发展,并停止反应与四个0.1 M PB洗。
    9. 倒出ABC过氧化物酶溶液并用PBS洗涤5分钟,3次。
    10. 将切片储存在0.05 M PB中。

  4. 免疫染色后连续安装
    1. 将第一个小瓶中的内容物用0.01M PB(在H 2 O中0.05M PB的1:5稀释液)倒入大培养皿中,并使用精细画笔排列在柱中。
    2. 仔细移动第二个小瓶的内容,并安排在第一个小瓶的列旁边的列。
    3. 重复,直到所有的小瓶被排列成彼此相邻的列。

    4. 使用画笔将相邻的连续切片放置在幻灯片上,逐行移动(图1和图2)
    5. 如果干式IHC切片,通过在ddH 2 O(在ddH 2 O中稀释1:4 0.5%w / v明胶)包被0.12%w / v明胶来制备载玻片,参见食谱)用一把中等尺寸的称重船的画笔。通过将磨砂侧面放在称重船的方形边缘上,并将水下的一侧放在喷口的底部斜面上(视频4),将半边玻璃留下。
    6. 将大片培养皿中的初始切片移入含有0.12%明胶的中等重量的船中,将切片置于溶液中,定位切片,然后小心地将切片滑到离滑片的浸没边缘的适当位置。
    7. 将滑块推向称重舟的喷口,从0.12%的明胶中暴露更多的载玻片,并在步骤D6中重复相邻切片的过程以填充载玻片的其余部分。
    8. 在Kimwipe上的干燥的滑动边缘上,在22°C空气干燥24小时,然后进行复染,脱水,固定介质和盖玻片的放置。

      视频4


      图2.使用连续切片将套管定位在腹侧被盖区(VTA)中。用含辣根过氧化物酶和3',3'-二氨基联苯胺(DAB)显色的抗酪氨酸羟化酶染色明胶嵌入的大鼠脑切片。将切片干燥固定在载玻片上,用0.02%硫堇复染3分钟,通过乙醇脱水系列并用Entellan固定培养基盖上盖子。六个80微米的连续切片顺序安装,从左上方沿水平方向移动到右下方,小瓶和切片编号标记在切片上方(请参见图1中小瓶和切片的来源)。

数据分析

使用Olympus CX22LED光学显微镜(图2)验证大鼠VTA中套管放置的定位。另外,通过使用Olympus CX22LED光,定性分析大鼠纹状体中立体定向注射的抗ChAT-皂草素抗体定向损伤(Aoki等人,2015,图3)的程度的验证和作图显微镜和解剖标志从老鼠大脑地图集(Paxinos和沃森,2004年)。


图3.抗体靶向免疫毒素递送至大鼠纹状体中的胆碱能神经元的验证和病变作图A.大鼠纹状体DAB染色的切片的代表性冠状切片显示大部分完整的NeuN染色,但是胆碱能细胞特异性缺失中间神经元在兔抗ChAT-皂草素介导的损伤病例(箭头背内侧层,DMS或腹侧纹状体,VS)中进行ChAT染色。比例尺= 1毫米。 B.使用非选择性山羊IgG-皂草素注射液作为对照(箭头)的由虚线区域(NeuN)表示的有限的非特异性损伤的实例,其中出现较少的细胞并且实例更广泛的非特异性缺失。 C和D.在每个行为条件中显示胆碱能神经元缺失的最受限制的(实心黑色)和最广泛的区域(灰色)。坐标距前囟切片的距离显示在左边。 DMS(C)和VS(D)中病变的程度在所有情况下都相似。 DAB染色照片中的解剖标志:LV,侧脑室; CC,胼体; AC,前连合。图经过修改以进行澄清并经过许可转载(Aoki等人,2015年)。

笔记

强烈建议小瓶和称量的小船在动物身份证前用手标记,并且小心地将大脑从一个容器转移到另一个容器,以确保不会混淆个人的身份。

食谱

  1. 4%PFA / 0.1M PB(4%多聚甲醛/0.1M磷酸盐缓冲液,pH7.4)
    在ddH 2 O中的4%w / v多聚甲醛 80mM Na 2 HPO 4 4/2 20 mM NaH 2 PO 4 4
  2. 0.1M PB(0.1M磷酸盐缓冲液,pH7.4)
    在ddH 2 O中的80mM Na 2 HPO 4: 20 mM NaH 2 PO 4 4
  3. 30%蔗糖/0.1M PB(30%蔗糖0.1M磷酸盐缓冲液,pH7.4)
    在ddH 2 O中30%w / v蔗糖 80mM Na 2 HPO 4 4/2 20 mM NaH 2 PO 4 4
  4. 4%多聚甲醛/ 10%蔗糖的0.1M磷酸盐缓冲液,pH7.4中 在ddH 2 O中的4%w / v多聚甲醛 20%w / v蔗糖
    80mM Na 2 HPO 4 4/2 20 mM NaH 2 PO 4 4
  5. 0.05M PB(0.05M磷酸盐缓冲液,pH7.4)
    在ddH 2 O中的40mM Na 2 HPO 4: 10 mM NaH 2 PO 4 4
  6. 30%蔗糖0.05M磷酸盐缓冲液,pH7.4
    在ddH 2 O中30%w / v蔗糖 40mM Na 2 HPO 4 4 10 mM NaH 2 PO 4 4
  7. 阻止解决方案
    1x PBS
    5%中学宿主血清
    0.2%Triton X-100
  8. 抗体解决方案
    1x PBS
    2%中学宿主血清
    0.2%Triton X-100
  9. 在ddH 2 O中0.5%w / v明胶 ddH 2 O至50℃
    0.5%w / v明胶

致谢

我们感谢Tom Ruigrok博士提供了明胶包埋的初始方案,Mayank Aggarwal允许我们在图2所示的VTA中显示套管放置的彩色玻片。我们也非常感谢FrançoisBeauchain进行批判性校正,Yumiko Akamine提供帮助与视频制作。最后,我们把我们的帽子给安德烈斯卡拉斯科,呼吁我们注意生物协议。
来自人类前沿科学计划(AWL,JRW)的支持,JSPS对挑战性探索性研究的资助,JSPS研究员的资助以及A类青年科学家的资助(SA)进行了研究在这个出版物可能。作者声明没有竞争的财务利益。

参考

  1. Aoki,S.,Liu,A.W。,Zucca,A.,Zucca,S.and Wickens,J.R。(2015)。 纹状体胆碱能中间神经元在大鼠移位中的作用 J Neurosci 35(25):9424-9431。
  2. Aquili,L.,Liu,A.W。,Shindou,M.,Shindou,T。和Wickens,J.R。(2014)。 在特定的时间段,伏隔核神经元的光遗传学抑制增加了行为的灵活性。 a> Learn Mem 21(4):223-231。
  3. Griffioen,H.A.,Van der Beek,E.和Boer,G.J。(1992)。 明胶包埋保存病灶损伤的下丘脑和脑室移植物,用于振动切片和免疫细胞化学。
    J Neurosci方法 43(1):43-47。
  4. Paxinos,G.和Watson,C。(2004)。 老鼠脑在立体坐标。
  5. Suzuki,L.,Coulon,P.,Sabel-Goedknegt,E.H。和Ruigrok,T.J。(2012)。 组织脑部投射到小鼠小脑后部小脑区。< J Neurosci 32(32):10854-10869。
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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: © 2017 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. Liu, A. W., Aoki, S. and Wickens, J. R. (2017). A Streamlined Method for the Preparation of Gelatin Embedded Brains and Simplified Organization of Sections for Serial Reconstructions. Bio-protocol 7(22): e2610. DOI: 10.21769/BioProtoc.2610.
  2. Aoki, S., Liu, A. W., Zucca, A., Zucca, S. and Wickens, J. R. (2015). Role of striatal cholinergic interneurons in set-shifting in the rat. J Neurosci 35(25): 9424-9431.
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