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Jan 2020
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Using the Parafilm-assisted Microdissection (PAM) Method to Sample Rodent Nucleus Accumbens
应用膜辅助显微切割(PAM)对啮齿类伏隔核进行采样   

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Abstract

Microdissection techniques are very important for anatomical and functional studies focused on neuroscience, where it is often necessary microdissect specific brain areas to perform molecular or anatomical analyses. The parafilm®-assisted microdissection (PAM) was previously described and involves the microdissection of tissue sections mounted on parafilm-covered glass slides. In this work, we describe the use of the PAM method to microdissect rodent nucleus accumbens (NAc). (1) We first describe the best way to perform the mouse euthanasia and how to remove the brain. (2) Next, we describe how to prepare the slides with parafilm® that will be used to receive the brain slices. (3) Following, we describe how to handle the brain in the cryostat, how to align the hemispheres and how to identify the NAc antero-posterior limits. (4) We also describe how to perform the staining and dehydration of the slices, a critical step to facilitate the microdissection and preserve macromolecules. (5) In the final step, we describe how to identify the dorso-ventral and latero-medial limits of the NAc and, finally, how to perform the manual microdissection of the area. This is a low-cost technique that allows the researcher to specifically microdissect any brain region, from which intact RNA and proteins can be extracted to perform several molecular analyses (e.g., real-time PCR, Western blot, and RNA-seq).

Keywords: Microdissection (显微解剖), Nucleus accumbens (伏隔核), Neuroanatomy (神经解剖学)

Background

Nucleus accumbens (NAc) is part of the basal ganglia located in the rostroventral part of the striatum. Specifically, NAc is part of a structure that we call ventral striatum, which also comprises the olfactory tubercle. It is well known the role of NAc in the dopaminergic mesolimbic pathway, which regulates motivational behavior and is responsible for emotional and contextual behaviors (Baik, 2013). NAc has already been implicated in psychiatric disorders (e.g., depression), drug addiction, obesity, and chronic pain, which show the relevance in studying the neurobiology of this brain structure (Nestler and Carlezon, 2006; Volkow et al., 2011; Volkow and Morales, 2015; Brandão et al., 2019; Serafini et al., 2020).

Based on this, it is important to have a precise technique describing how to microdissect the NAc, sampling this structure for molecular analysis. The punch technique (Palkovits, 1983), for example, allow us to microdissect brain sections (including NAc) with a hollow needle and is widely used in neurobiological laboratories. Advantages of punch technique are low cost and fast sampling. Disadvantage of using this technique is the limited accuracy, once the experimenter is restricted to the cylindrical shape of the punch tool and because the experimenter must punch fresh tissue, which is hard to identify some surrounding neuroanatomic structures of the NAc, such as the ventral pallidum.

Because of this, depending on the molecular analysis and the objective, it is preferable using a more accurate technique, such as laser-assisted microdissection (or laser capture microdissection). Laser-assisted microdissection technique is a method to microdissect tissue under direct microscopic visualization (Espina et al., 2006). Advantages of using this technique are the high accuracy on delineation of the NAc and the capacity of sampling smaller areas (e.g., NAc core or shell) or even cell populations. Disadvantages of this technique are the long time until sampling the tissue and the high cost to perform, requiring an entire facility with microscope and laser equipment.

Interestingly, Franck et al. (2013) described the parafilm-assisted microdissection (PAM) as a sampling method, a technique which involves the microdissection of tissue sections mounted on parafilm-covered glass slides. This group described the application of this technique for prostate cancer (Quanico et al., 2015), ovarian tissue (Delcourt et al., 2017) and for specific brain areas such as cerebellum, hypothalamus, and hippocampus (Franck et al., 2013; Quanico et al., 2017a and 2017b; Delcourt et al., 2018). The PAM technique can be easily performed, once it uses low-cost materials and allows high accuracy on delineating the target brain area. Disadvantage of this technique is the limitation in microdissection smaller structures, such as subthalamic nucleus, for example.

In this manuscript, we will describe the whole process, from the brain storage after euthanasia to molecular analysis, of using the PAM technique to specifically microdissect the rodent NAc, also describing how to stain the brain slices to easily identify the anatomical limits of the NAc. It is worth mentioning that, as above-mentioned, this protocol can be used to microdissect a great range of brain regions, depending on the size and shape. Tissue obtained from this technique can be used for molecular analyzes such as real-time PCR, Western Blot, and RNA-seq.

Materials and Reagents

  1. Common microscope slides 25 mm x 75 mm (e.g., Sigma, catalog number: S8902)

  2. Plastic paraffin or laboratory film (Parafilm®, Bemis Company, catalog number: P7543)

  3. Cryostat blade (e.g., Leica, catalog number: 14035838383)

  4. Scalpel n° 11 (e.g., Sigma, catalog number: S2771)

  5. Adhesive tape (e.g., 3M, catalog number: 2214)

  6. Aluminum foil (e.g., Sigma, catalog number: Z185140)

  7. Staining racks (e.g., Electron Microscopy Science, catalog number: 70315)

  8. 50 ml conical tube (e.g., Fisher Scientific, catalog number: 14-432-22)

  9. Needle 18G (e.g., GIME Professional Medical Products, catalog number: 23750)

  10. 1.5 ml microtube (e.g., Sigma, Eppendorf, catalog number: Z606340; preferably RNAse free)

  11. Tissue freezing medium (e.g., Tissue-Tek® O.C.T. Compound, Sakura, catalog number: 4583)

  12. Ethanol 100% (room temperature -18 °C to 25 °C) (e.g., Sigma, catalog number: E7023)

  13. Ethanol 100% (4 °C) (e.g., Sigma, catalog number: E7023)

  14. Cresyl violet (e.g., Sigma, catalog number: C5042)

  15. Methylbuthane (e.g., Sigma, catalog number: 277258) – room temperature

  16. Dry ice

  17. Cool box

  18. Metallic can

  19. 2 solutions of Ethanol 70% (4 °C; diluted from ethanol 100%) (e.g., Sigma, catalog number: E7023; see Recipes)

  20. Ethanol 50% (4 °C; diluted from ethanol 100%) (e.g., Sigma, catalog number: E7023; see Recipes)

  21. Cresyl Violet 1% (see Recipes)

Equipment

  1. Cryostat (e.g., Leica, model: CM1950)

  2. Tissue holder or cryostat chucks (e.g., MarketLab, PolarChuckTM, catalog number: ML39130)

  3. (Optional) Lupe (e.g., ZEISS, model: Stemi 508)

Procedure

  1. Euthanizing the mouse and handling the brain

    1. It is recommended to euthanize the mouse through guillotine decapitation or cervical dislocation, being careful to not damage the brain. Chemical techniques (both injectable and inhalational) of euthanasia are not indicated because they can cause biochemical changes in the brain (Leary et al., 2013), such as isoflurane even at low doses (Bekhbat et al., 2016). However, which euthanasia technique to be used is the researcher’s choice, although the above-mentioned arguments must be considered.

    2. Remove the brain from the skull using the technique described by Spijker (2011) or any other of your choice. It is important to remove fast and with no damage to the tissue.

    3. Freeze the brain immersing it in methylbutane at -50 °C (using dry ice).

      CRITICAL STEP: For this, you can put dry ice in a cool box and the methylbutane in a metallic recipient (e.g. metallic can). Place the methylbutane-containing metallic recipient on the dry ice to cool down. It's important to mention that the rapid freezing of the brain, which happens in temperatures below -50 °C, may cause cracks in the tissue when slicing in the cryostat. Therefore, keep the methylbutane temperature above -50 °C (you can use an infrared thermometer for greater accuracy) during the procedure.

    4. After freezing the brain, it is important to store it individually in the freezer at -80 °C in a package that will preserve the organ structure. You can store the brain at -80 °C up to one year (we did not test more than this period in our laboratory). In our laboratory we use low cost materials to stock brain, such as that shown in Figure 1 which is made with one-layer aluminum foil and adhesive tape.



      Figure 1. Stocking the brain. A. Front view of the package to stock the brain in the freezer (-80 °C), produced in aluminum foil and adhesive tape. B. Upper view of the package to stock the brain in the freezer, note the top opening to place the brain inside the package. C. After placing the brain, seal the package by folding tightly the top of the aluminum foil.


  2. Preparing the glass slides

    1. Cut pieces of Parafilm® that fits to cover the glass slide surface (25 mm x 75 mm).

      Note: We recommend cutting the Parafilm® slightly smaller than the glass slide, this way is easier to perform the staining using the staining racks.

    2. Attach the Parafilm® to the glass slide using adhesive tape, leaving free the surface on which the slices of the brain will be positioned (Figure 2).

      Note: Do not stretch the piece of Parafilm®.



      Figure 2. Microscope slide prepared to PAM protocol. Detail of the microscopy slide (1) covered with a not stretched small piece of Parafilm® (2) fixed with adhesive tape (3).


  3. Processing the brain in the cryostat

    1. Place the brain in the tissue holder with the anterior region facing upwards (Figure 3), that is, immersing in the tissue freezing medium the posterior side of the brain (i.e., the side containing the medulla oblongata). This way it will be easier to find reference points to align the hemispheres.

      CRITICAL STEP: If you need to microdissect structures located in the brainstem is recommended positioning the brain in the tissue holder inversely, immersing the anterior region of the brain (i.e., the side containing the olfactory bulb) in the tissue freezing medium. This is necessary because the way the brain is frozen (using methyl butane at -50 °C) makes the brain stem fragile, breaking easily due to the pressure of the cryostat blade.

      Note: It is also recommended positioning the brain vertically in the holder of the tissue holder, this also prevents the brainstem from breaking (see Figure 3C).



      Figure 3. Placing the brain in the cryostat apparatus. A. Anatomical directions of the mouse brain in a top view (scale in cm). B. Placement of the brain in the tissue holder (or cryostat chucks), note the anterior region directed upwards (C).

      Note: How we recommend placing the brain in the holder of the tissue holder: vertically, hampering the brainstem break due to the pressure of the cryostat blade.


    2. Align the hemispheres so the cryostat blade will cut the brain perpendicularly. To align the two hemispheres (when they are both in the same position - or very similar - in relation to bregma) of the brain the experimenter must slice the brain until it is divided into four quadrants, separated by the rhinal sulcus and longitudinal fissure (see Figure 4). The experimenter should use these depressions as neuroanatomic reference to align the hemispheres, by moving the holder of the tissue holder.

      CRITICAL STEP: During slicing, maintain the cryostat temperature at -15 °C, above this temperature the slice will unfreeze, and below this temperature the slice will crack.



      Figure 4. Neuroanatomic references to align the hemispheres. Note that in the image (A) the hemispheres are not aligned, and, in the image (B), the hemispheres are aligned. C. Insert showing a graphical scheme of the neuroanatomic references (rhinal sulcus (rs) = black arrow; longitudinal fissure (lf) = green arrow).


    3. Identify the beginning of the NAc and start collecting the slices on the slide with Parafilm®. The NAc begins when the experimenter is able to identify the corpus callosum and anterior commissure by the naked eye (see Figure 5A). From this moment, the experimenter should place the slices in one of the slides prepared with Parafilm® (see an important hint on how to perform this step in Figure 6) until the anterior commissure cross the median plane of the brain (commissurated region, where there is passage of fibers from one hemisphere to another), as shown in Figure 5B. This commissurated region indicates the NAc posterior limit. Carefully place the slices on the top of the Parafilm® covering the slide. After collecting all the slices from the brain, the slide should look like the one shown in Figure 7. If you slice with a thickness of 60µm you will collect approximately 24 slices until you see the anterior commissure crossing the median plane of the brain.

      CRITICAL STEP: After placing the slices on the slide do not remove from the cryostat until start the next step (staining and dehydration), otherwise the slices will unfreeze.

      Note: If it is difficult to visualize by naked eye, the experimenter can collect a slice on a common slide (with no Parafilm®), stain it with a drop of Cresyl Violet 1% (diluted in 50% alcohol) and visualize in a microscope.



      Figure 5. Identifying anterior and posteriors limits of the NAc. A. Reference points to identify the beginning of the NAc (anterior anatomical limit) and start collecting slices in the slides prepared with Parafilm®. B. Reference points to locate the end of the NAc (posterior anatomical limit) and stop collecting slices, note the commissurated region. C. Insert showing a graphical scheme of the neuroanatomic references (corpus callosum (cc) = green arrow; anterior commissure (aco) = black arrow; lateral ventricle (lv) = blue arrow).



      Figure 6. How to position the slices on the slide with Parafilm®. First, warm the back of the slide (which has no Parafilm®) with your finger (A) then approach the slide to the slice, which will attach to the Parafilm® (B).



      Figure 7. Slide with NAc slices. Detail of the slide with the NAc slices placed on the top of the Parafilm®.


  4. Staining the slices

    1. Stain and dehydrate the slices (which facilitates microdissection and preserves macromolecules), using the bath sequence described below, by immersing the slices-containing slides into the staining racks. It is worth mentioning that the slices do not fall from the slide after staining, once the Parafilm® helps in the slice adhesion. After staining the slices, the slide should look like the one shown in Figure 8.

      1. 120 s in alcohol 70% (4 °C);

      2. 90 s in Cresyl Violet 1% (diluted in 50% alcohol) (4 °C);

      3. 2x Fast wash (2s) in alcohol 70% (4 °C);

      4. 120 s in 100% alcohol (4 °C);

      5. 120 s in 100% alcohol (room temperature);

      6. Leave to dry for 15 min (optional: use a lab hood).

      CRITICAL STEP: Renew all alcoholic solutions after 20 uses or after 30 days of the first use. This does not apply to Cresyl Violet 1% solution, which can be used up to 60 times or after 3 months of the first use.



    2. Figure 8. Slide with stained NAc slices. Detail of the slide with stained NAc slices placed on the top of the Parafilm®.


    3. Stock the slides in pairs, one with the back to another, inside a 50 ml conical tube (Figure 9).

      CRITICAL STEP: To preserve tissue dehydration, seal the conical tube with Parafilm®. This way the slide with the slices can be frozen at -20 °C for several months with no damage to the tissue.



      Figure 9. Stocking the slides. Stock the slides in pairs inside a 50 ml conical tube, one with the back to another.


  5. NAc manual microdissection

    1. Remove the conical tubes containing the slides from the freezer and left at room temperature for, at least, 15 min to unfreeze.

      CRITICAL STEP: Just open the conical tube after total thawing.

    2. Position the slide under a magnifying glass (optional), which can facilitate to visualize the brain areas. At this point, the experimenter must identify the anterior commissure (a whitish circular structure). Assisted by a scalpel n° 11 scrape all the tissue belonging to the anterior commissure, removing it. After performing this scraping, make a cut around the NAc (also cutting the Parafilm®). At this moment we recommend the experimenter to consult an atlas (e.g., Franklin and Paxinos, 2013), confirming that only NAc tissue is being removed. Figure 10 shows in sequence the location of the NAc and how the cuts around the NAc should be performed using the scalpel. After this step, remove the tissues with assistance of a needle, placing it in a 1.5 ml microtube (see Figure 11). Seal the tube with Parafilm® and store it at -80 °C for molecular biology procedures.

      Note: To facilitate the NAc removal with the needle, the experimenter can leave a slightly raised portion of the Parafilm® when cutting with the scalpel.



      Figure 10. NAc manual microdissection. (A1), (B1), and (C1) show the representative sequence of the beginning (anterior limit), middle and end portion (posterior limit) of the NAc, respectively. (A2), (B2), and (C2) show these same representative slices after NAc been cutted by a scalpel (note that NAc is removed along with the Parafilm®). (A3), (B3), (C3), and (D2) show a graphical scheme of the neuroanatomic references. (D1) shows a slice without NAc, note that the anterior commissure is crossing the median plane of the brain, indicating the end of the NAc. (corpus callosum (cc) = green arrow; anterior commissure (aco) = black arrow; lateral ventricle (lv) = blue arrow; dorsal striatum yellow arrow).

      Note: In most of the slices you can consider the superior limit of the NAc as the dorsal striatum, inferior limit as ventral pallidum, medial limit as medial septal complex and the end of the lateral ventricle. Lateral limit is harder to identify, but you can consider the beginning of the cortex.



      Figure 11. Using microtubes to stock NAc tissue. 1.5 ml microtube used to stock NAc tissue taken from the mice's brain along with the Parafilm®.

Data analysis

Microdissected tissue can be processed with TRIzol® reagent for the macromolecules extraction. Microdissection can yield low amount of tissue, because of this, we recommend using 0.5 ml of TRIzol® reagent per sample. For the other reagents used in the TRIzol® reagent protocol, we recommend using the proportions suggested in the reagent data sheet (see TRIzol® Reagent User Guide here).

Note: The pieces of Parafilm® remain on the surface of the aqueous phase after the first centrifugation of the protocol (phase separation), at this moment they can be easily removed.

    In the next steps, some changes in the TRIzol® reagent data sheet can be performed. In the RNA precipitation step, it is recommended to add to the aqueous phase, besides 0.25 ml of 100% isopropanol, 5 µg of RNase-free glycogen or 2 µl of linear acrylamide as carriers (they will co-precipitate with the RNA, increasing the extraction efficiency). In this RNA precipitation step, we also recommend incubating the sample at -20 °C overnight (to maximize RNA precipitation) and then centrifuging at 12,000 x g for 10 min. In the next step of the protocol (RNA wash), we recommend performing one or two RNA washes, more than this may cause excessive RNA loss. Finally, the RNA can be resuspended using 10 μl of RNase-free water or 0.5% SDS solution.

    It is important to mention that TRIzol® reagent protocol may not have high proteins extraction efficiency. Therefore, we suggest an alternative protein extraction protocol using 8 M urea. For this suggested protocol, add 500 μl of 8 M urea to the microdissected sample, incubate for 20 min at room temperature (the experimenter can perform 3 fast pulses in a sonicator), and then centrifuge at 12,000 x g for 5 min. Use the supernatant. The pieces of Parafilm® will also be suspended after this centrifugation and can be easily removed.


  1. RNA analyses

    The RNA extracted by TRIzol® reagent protocol yields material in quality and quantity sufficient for real-time PCR and RNA-seq techniques. These techniques have already been successfully performed in our laboratory with samples obtained from the PAM protocol (see Pagliusi Jr. et al., 2018 and Brandão et al., 2019). Table 1 shows the RNA quantification and purity of 5 different samples microdissected through the PAM protocol and processed using above-mentioned TRIzol® reagent protocol.


  2. Table 1. RNA analyses. RNA quantification and purity of 5 different NAc samples microdissected through the PAM protocol and processed using the TRIzol® reagent protocol.


  3. Protein analyses
    Although we believe that the protein extracted by the TRIzol® reagent protocol yields material in quality and quantity sufficient for Western-Blot (WB) and ELISA techniques (in samples microdissected through the PAM protocol), only WB was tested in our laboratory. Also, Franck et al. (2013) performed mass spectrometry using samples from the PAM protocol. Figure 12 shows a WB for β-actin and a ponceau performed in our laboratory using 5 different samples microdissected through the PAM protocol (same samples used for the above-mentioned RNA analyses) and processed using the TRIzol® reagent protocol.



    Figure 12. Protein analyses. (A) Western blot for β-actin and (B) ponceau staining membrane performed in our laboratory using NAc samples from the PAM protocol and processed using the TRIzol® reagent protocol.

Recipes

  1. Cresyl Violet 1% (stock temperature: 4 °C)

    2.5 g Cresyl violet

    250 ml ethanol 50%

  2. Ethanol 70% (stock temperature: 4 °C)

    60 ml ethanol 100%

    140 ml distilled water

  3. Ethanol 50% (for Cresyl Violet 1% preparation):

    100 ml ethanol 100%

    100 ml distilled water

Acknowledgments

This study was financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 and São Paulo Research Foundation (FAPESP) - grant number 2013/09749-4 and 2015/26777-7. We are grateful for the previous studies ( Franck et al., 2013 ; Quanico et al., 2015 , 2017a and 2017b; Delcourt et al., 2017 and 2018), which allowed us to perform and describe the protocol for NAc microdissection. We also thanks the artist Flávia Pagliusi (for contact flaviapgl@gmail.com) that made the graphical schemes for Figures 4, 5 and 10.

Competing interests

We declare no competing interests.

Ethics

All experiments presented here were approved by the Ethics Committee on the Use of Animals at the Biology Institute of the University of Campinas - CEUA/UNICAMP (protocols 3849- 1 and 4249-1).

References

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简介

[摘要]显微解剖技术对于专注于神经科学的解剖学和功能研究非常重要,在显微学中,通常有必要对特定的大脑区域进行显微解剖以进行分子或解剖学分析。石蜡膜® -assisted显微切割(PAM)先前描述的,并且涉及安装在封口膜覆盖的载玻片上的组织切片的显微切割。在这项工作中,我们描述了使用PAM方法微分散啮齿动物伏隔核(NAc)。(1)我们首先描述执行小鼠安乐死的最佳方法以及如何去除大脑。(2)接下来,我们介绍如何用石蜡制作的幻灯片® 那将被用来接收大脑切片。(3)接下来,我们描述如何在低温恒温器中处理大脑,如何对齐半球以及如何识别NAc前后限。(4)我们还描述了如何对切片进行染色和脱水,这是促进显微切割和保存大分子的关键步骤。(5)在最后一步,我们描述了如何识别NAc的背腹和后内侧界限,以及最后如何进行该区域的手动显微解剖。这是一种低成本的技术,该技术允许研究者具体microdissect任何脑区域,从该完整的RNA和蛋白质可被提取为执行几个分子分析(例如。,实时PCR,西方印迹和RNA-SEQ)。

[背景]伏隔核(NAC)是位于纹状体的rostroventral部分基底节的一部分。具体而言,NAc是我们称为腹侧纹状体的结构的一部分,该结构也包含嗅觉结节。众所周知,NAc在多巴胺能中脑边缘途径中的作用,其调节动机行为并对情绪和情境行为负责(Baik,2013)。伏隔核已经被牵连的精神疾病(例如,抑郁症),药物成瘾,肥胖,慢性疼痛,这表明在研究这个大脑结构的神经生物学的相关性(内斯特勒和Carlezon,2006;沃尔科夫等,2011;沃尔科夫和Morales,2015;Brandão等,2019; Serafini等,2020)。

基于此,拥有一种精确的技术来描述如何对NAc进行显微解剖,对该结构进行采样以进行分子分析非常重要。例如,打孔技术(Palkovits,1983年)使我们可以用空心针显微解剖大脑切片(包括NAc),并广泛用于神经生物学实验室。打孔技术的优点是低成本和快速采样。一旦将实验人员限制在打孔工具的圆柱形状中并且由于实验人员必须打孔新鲜的组织,使用此技术的准确性会受到限制,这很难确定NAc周围的一些神经解剖结构,例如腹侧苍白球。

因此,取决于分子分析和目的,优选使用更精确的技术,例如激光辅助显微切割(或激光捕获显微切割)。激光辅助显微切割技术是一种在直接显微镜观察下显微切割组织的方法(Espina等,2006)。使用该技术的优点是在描绘NAc时具有很高的准确性,并且可以对较小区域(例如,NAc核心或外壳)甚至细胞群进行采样。该技术的缺点是采样组织之前需要很长时间,并且执行成本很高,因此需要配备显微镜和激光设备的整个设备。

有趣的是,Franck等。(2013年)描述了膜旁辅助显微解剖(PAM)作为一种采样方法,该技术涉及对安装在覆盖有膜下的载玻片上的组织切片进行显微解剖。该小组描述了该技术在前列腺癌(Quanico等人,2015),卵巢组织(Delcourt等人,2017)以及特定大脑区域(如小脑,下丘脑和海马体)中的应用(Franck等人,2013) ; Quanico等人,2017a和2017b; Delcourt等人,2018)。PAM技术一旦使用低成本材料即可轻松实现,并在描绘目标大脑区域时具有很高的准确性。该技术的缺点是在显微解剖中限制了较小的结构,例如丘脑下核。

在本文中,我们将描述从安乐死后的大脑存储到分子分析的整个过程,其中包括使用PAM技术对啮齿动物NAc进行专门的显微解剖,还描述了如何对脑切片进行染色以轻松确定NAc的解剖学界限。值得一提的是,如上所述,该协议可用于根据大小和形状对大范围的大脑区域进行显微解剖。从该技术获得的组织可用于分子分析,例如实时PCR,Western Blot和RNA-seq。

关键字:显微解剖, 伏隔核, 神经解剖学

 
材料和试剂
 
1.普通的显微镜载玻片25毫米×75毫米(例如,Sigma,目录号:S8902) 
2.塑料石蜡或实验室膜(石蜡膜® ,比米斯公司,目录号:P7543) 
3.低温恒温器刀片(例如,莱卡,目录号:14035838383) 
4.手术刀Ñ ° 11(例如,Sigma,目录号:S2771) 
5.胶带(例如,3M,目录号:2214) 
6.铝箔(例如,Sigma,目录号:Z185140) 
7.染色架(例如,电子显微镜科学,目录号:70315) 
8. 50 ml锥形管(例如,Fisher Scientific,目录号:14-432-22) 
9针18G(例如,吉美专业医疗产品,目录号:23750) 
10. 1.5毫升微管(例如,Sigma公司,的Eppendorf,目录号:Z606340;优选RNA酶免费)   
11.组织冷冻介质(例如,组织-Tek的® OCT化合物,樱,目录号:4583 )                 
12.乙醇100%(室温- 18 ℃,至25 ℃下;例如,Sigma,目录号:E7023)   
13. 100%乙醇(4 °C ;例如,Sigma,目录号:E7023 )   
14.甲酚紫(例如,Sigma,目录号:C5042)   
15.甲基丁烷(例如,Sigma,目录号:277258)–室温   
16.干冰   
17.酷箱   
18.金属罐   
19. 2种浓度为70%的乙醇溶液(4 °C ;用100%乙醇稀释;例如,Sigma,目录号:E7023;请参见食谱)   
20. 50%乙醇(4 °C ;从100%乙醇中稀释;例如,Sigma,目录号:E7023;请参见食谱)   
21.甲酚紫1%(请参阅食谱)   
 
设备
 
低温恒温器(例如,徕卡,莫德尔:CM1950 )
组织保持器或低温恒温器的卡盘(例如,MarketLab ,PolarChuck TM ,目录号:ML39130)
(ø ptional)卢佩(例如,ZEISS,型号:STEMI 508 )
 
程序
 
安乐死老鼠并处理大脑
建议通过断头台斩首或颈椎脱臼来安乐死小鼠,注意不要损伤大脑。未标明安乐死的化学技术(注射和吸入),因为即使在低剂量下,它们也会引起大脑的生化变化(Leary等人,2013),例如异氟烷(Bekhbat等人,2016)。然而,要使用哪个安乐死技术是研究者“的选择,虽然上述参数必须考虑。
使用Spijker(2011)描述的技术或您选择的任何其他方法,从头骨上取下大脑。重要的是要快速去除并且对组织没有损坏。
冻结大脑,将其浸入-50 °C的甲基丁烷中(使用干冰)。
关键步骤:为此,您可以将干冰放在凉爽的盒子中,将甲基丁烷放在金属容器(例如金属罐)中。将含甲基丁烷的金属容器放在干冰上冷却。值得一提的是,在低温恒温器中切片时,在低于-50 °C的温度下发生的大脑快速冻结可能会导致组织破裂。因此,在操作过程中,应使甲基丁烷温度保持在-50 °C以上(可以使用红外温度计以提高准确性)。
冷冻大脑后,重要的是将其单独保存在-80 °C的冰箱中,并保存在能够保存器官结构的包装中。您可以将大脑储存在-80 °C的温度下长达一年(我们在实验室中进行的测试未超过此时间)。在我们的实验室中,我们使用低成本的材料来储存大脑,如图1所示,它是由一层铝箔和胶带制成的。
 


图1.储存大脑。一。该包装的正视图,用于将大脑储存在冰箱中(-80 °C ),由铝箔和胶带制成。乙。要在冰箱中存放大脑的包装上方视图,请注意顶部开口以将大脑放入包装内。Ç 。放置大脑后,通过紧密折叠铝箔的顶部来密封包装。
 
准备载玻片
裁片封口膜®适合以覆盖载玻片表面(为25mm× 75毫米)。
注:建议切割封口膜®比载玻片略小,这样更容易使用的染色架进行染色。
附加封口膜®使用粘合带的载玻片上,留下游离的在其上的脑切片将被定位在所述表面(图2 )。
注:d O不可拉伸一块封口膜® 。
 


图2.按照PAM协议准备的显微镜载玻片。所述显微镜载玻片的细节(1)覆盖有未延伸小块封口膜®用胶布固定(3)(2)。
 
              在低温恒温器中处理大脑
放置在组织保持架的脑与朝上的前部区域(˚F igure 3 ),即,在组织冷冻介质中的大脑的后侧浸渍(即,含有延髓的一侧)。这样,将更容易找到参考点以对齐半球。
关键步骤:我F你需要位于脑干microdissect结构建议在组织保持架定位大脑成反比,浸渍脑的前部区域(即,含有嗅球的一侧)在组织冷冻介质中。这是必要的,因为大脑的冷冻方式(在-50 °C下使用甲基丁烷)会使大脑的茎变得脆弱,由于低温恒温器刀片的压力而容易断裂。
注意:我吨还建议在组织保持器的保持器垂直定位大脑,这还防止从破脑干(参见图3 Ç )。
 


图3.将大脑放在低温恒温器中。A.在俯视图中(以厘米为单位)的鼠标大脑的解剖学方向。B.将大脑放置在组织固定器(或低温恒温器卡盘)中,注意前部区域向上(C)。
注意:我们建议如何将大脑放置在组织支架的支架中:垂直放置,以防止由于低温恒温器刀片的压力而引起的脑干破裂。
 
对齐半球,这样低温恒温器刀片将垂直切开大脑。为了对齐两个半球(当它们相对于前relation处于相同位置或非常相似时),实验人员必须将大脑切成薄片,直到将其分为四个象限,并由鼻沟和纵向裂隙分开(见˚F igure 4 )。实验者应通过移动组织固定器的固定器,将这些凹陷作为神经解剖学参考来对齐半球。
关键步骤:d uring切片,保持在-15低温恒温器温度℃的,高于此温度的切片将解冻,并在该温度以下的切片会开裂。
 


图4.对齐半球的神经解剖学参考。注意,在图像(A)中,半球未对齐,而在图像(B)中,半球未对齐。C.插入物显示神经解剖参考的图形方案(鼻沟(rs )=黑色箭头;纵裂(lf )=绿色箭头)。
 
确定南汽开始,并开始收集与封口膜幻灯片片® 。在NAC开始时实验者能够识别胼胝体和通过肉眼前连合(参见˚F igure 5A )。从这一刻起,实验者应放置在一个幻灯片与准备切片封口膜® (参阅有关如何执行此步骤中的一个重要的暗示˚F igure 6 ),直到前连合交叉大脑的中间平面(commissurated区域,其中存在从一个半球到另一纤维的通道),如图˚F igure 5B 。该接合区域指示NAc后极限。小心地放在封口膜顶部切片®覆盖的幻灯片。从大脑收集所有切片后,将载玻片应该像所示的一个˚F igure 7 。如果切片的厚度为60μm,您将收集大约24片切片,直到看到前连合穿过大脑的正中平面为止。
关键步骤:在将切片放在载玻片上之前,切勿从低温恒温器中取出切片,直到开始下一步(染色和脱水),否则切片会解冻。
注意:我的f,难以通过肉眼来可视化,实验者可以收集在一个共同的载玻片上的切片(无封口膜® ),用甲酚紫1%(在50%乙醇中稀释)的下降留下污点和可视化显微镜。
 


图5.确定NAc的前部和后部界限。A.参考点来识别伏隔核(前解剖限制)的开始,并开始收集在载玻片切片用Parafilm制备® 。B.定位NAc的末端(后解剖极限)并停止收集切片的参考点,注意接合区域。C.插入物显示神经解剖学参考的图形方案(call体(cc)=绿色箭头;前连合(aco )=黑色箭头;侧脑室(lv)=蓝色箭头)。
 


图6.如何定位与封口膜幻灯片片® 。首先,温滑的背面(没有封口膜®用手指)(A)则接近滑动片,这将附加到封口膜® (B) 。
 


图7.带有NAc切片的幻灯片。与NAC滑动的细节切片放置在封口膜的顶部® 。
 
              染色切片
通过将包含切片的载玻片浸入染色架中,使用以下所述的沐浴程序对切片进行染色和脱水(有助于显微解剖并保留大分子)。值得一提的是,片不从片染色,一旦封口膜后回落®切片粘连帮助。将切片染色后,载玻片应如图8所示。
酒精中120秒70%(4 °C);
在1%的Cresyl Violet中90 s(用50%酒精稀释)(4 °C )
在70%酒精(4 °C )中快速洗涤(2s)2次;
在100%酒精中(4 °C )120秒;
在100%酒精中放置120秒(室温);
晾干15分钟(可选:使用实验室通风橱)。
关键的一步:[R enew所有醇溶液后,使用20或后第一次使用30天。这不适用于1%的Cresyl Violet溶液,该溶液最多可使用60次或在首次使用3个月后使用。
 


图8.带有染色的NAc切片的幻灯片。与染色的伏隔滑动的细节切片放置在封口膜的顶部® 。
 
股票的滑动对,一个与回另一个,50ml锥形管内(˚F igure 9 )。
关键步骤:Ť ö保存组织脱水,密封锥形管用Parafilm ® 。这样,带有切片的载玻片可以在-20 °C下冷冻几个月,而不会损坏组织。
 


图9.存放幻灯片。将玻片成对放入一个50 ml的锥形管中,一个放回另一个。
 
              NAc手动显微解剖
从冷冻机中取出装有载玻片的锥形管,并在室温下放置至少15分钟以解冻。
关键步骤:Ĵ UST总解冻后打开锥形管中。
将幻灯片放在放大镜下(可选),以方便可视化大脑区域。在这一点上,实验者必须确定前连合(发白的圆形结构)。在n ° 11的手术刀的帮助下,刮除属于前连合的所有组织,并将其除去。执行此经过刮,使周围的伏隔核(也切割石蜡膜上的切割® )。在这个时刻,我们建议实验者咨询地图集(如。,富兰克林和Paxinos,2013) ,确认只有伏隔核组织被除去。图10依次显示了NAc的位置以及如何使用手术刀在NAc周围进行切割。在该步骤之后,用针的帮助去除组织中,将其放置在一个1.5毫升微型管(参见˚F igure 11 )。密封与管封口膜®并将其在-80存储℃下进行分子生物学过程。
注意:Ť华氏度acilitate NAc中去除与针,实验者可以离开的略微凸起部分的Parafilm ®用手术刀切割时。
 


图10. NAc手动显微解剖。(A1) ,(B1) ,和(C1)示出了伏隔的开始(前极限),中部和端部(后限制)的代表性序列,分别。(A2) ,(B2) ,和(C2)示出了这些相同的代表切片后伏隔被板缺由手术刀(注意,伏隔核与封口膜一起除去® )。(A3),(B3),(C3)和(D2)显示了神经解剖参考的图形方案。(D1)显示了没有NAc的切片,请注意,前连合横穿大脑的正中平面,表明NAc的末端。(corp体(cc)=绿色箭头;前连合(aco )=黑色箭头;侧脑室(lv)=蓝色箭头;纹状体背侧黄色箭头)。
注:我与N个最切片的话,可以考虑NAC的对扶优限为背侧纹状体,下极限,腹侧苍白球,内侧极限内侧隔复杂和侧脑室结束。横向界限较难辨认,但您可以考虑皮质的起点。
 


图11.使用微管储存NAc组织。1.5毫升微管用于从大脑mice`s与封口膜沿截取股票伏隔组织® 。
 
数据分析
 
显微组织可以用TRIzol处理®试剂为大分子萃取。显微切割可产生组织的低量,因为这一点,我们建议使用0.5毫升的TRIzol的® REAG每个样品ENT。对于在使用的TRIzol其它试剂®试剂协议中,我们建议使用在试剂数据片所建议的比例(见的TRIzol ®试剂用户指南这里)。
注:Parafil的片米®保持协议(相分离)的所述第一离心分离后,水相的表面上,在这一刻,他们可以容易地除去。
                在接下来的步骤,在TRIzol试剂一些变化®试剂数据表可以被执行。在RNA沉淀步骤中,建议将0.25 ml 100%异丙醇,5 µg不含RNase的糖原或2 µl线性丙烯酰胺作为载体添加到水相中(它们将与RNA共沉淀,从而增加提取效率)。在此RNA沉淀步骤中,我们还建议将样品在-20 °C下孵育过夜(以最大化RNA沉淀),然后以12,000 xg离心10分钟。在实验方案的下一步(RNA洗涤)中,我们建议进行一两次RNA洗涤,否则可能会导致RNA过多流失。最后,可以使用10μl不含RNase的水或0.5%SDS溶液重悬RNA。
                重要的是要提到的TRIzol是重要®试剂协议可能不具有高的蛋白质提取效率。因此,我们建议使用8 M尿素的另一种蛋白质提取方案。对于此建议的方案,加入500微升8M尿素到显微切割样品,温育在室温下(实验者可以执行在超声波仪3个快速脉冲)20分钟,然后以12,000rpm离心XG 5分钟。使用上清液。的碎片的Parafilm ®将人所以后暂停该离心分离,并且可以容易地除去。
 
RNA分析
              通过的TRIzol提取的RNA ®试剂协议产量在质量和数量足够的材料实时PCR和RNA-seq的技术。这些技术已经在我们的实验室中成功地使用了从PAM协议中获得的样品进行了实施(参见Pagliusi Jr.等人,2018和Brandão等人,2019 )。表1示出了RNA定量和通过PAM协议显微切割5个不同的样品纯度,并使用上述的TRIzol处理®试剂协议。
 
表1. RNA分析。RNA定量和通过PAM协议显微切割和使用的TRIzol处理5个不同伏隔样品纯度®试剂协议。
样品
定量ng / µl
比率260/280
1个
700.01
1.971
2
796.67
1.864
3
770.84
1.903
4
649.02
1.818
5
859.31
1.905
 
蛋白质分析
              虽然我们认为,受的TRIzol提取蛋白质®试剂协议收益率在质量和数量足够的蛋白质印迹(WB)和ELISA技术(在通过PAM协议显微切割样品)的材料,只有WB是在我们的实验室测试。另外,Franck等。(2013年)使用来自PAM协议的样本进行了质谱分析。图12 (使用相同的样品对上述RNA分析)示出了用于WBβ肌动蛋白和丽春在使用通过PAM协议显微切割5个不同的样品我们的实验室中进行,并使用的TRIzol处理®试剂协议。
 


图12.蛋白质分析。(A)对于β肌动蛋白和Western印迹(B)丽春红染色膜中使用伏隔采样来自PAM协议我们的实验室中进行,并使用的TRIzol处理®试剂协议。
 
菜谱
 
甲酚紫1%(原料温度:4 °C )
2.5克甲酚紫
250毫升乙醇50%
                乙醇70%(原料温度:4 °C )
60毫升乙醇100%
140毫升蒸馏水
                乙醇50%(用于1%的甲酚紫):
100毫升乙醇100%
100毫升蒸馏水
 
致谢
 
这项研究是由资金Coordenação德Aperfeiçoamento德Pessoal去NIVEL高级-巴西(CAPES) -财务代码001和圣保罗研究基金会(FAPESP) -允许数量2013 / 09749-4和2015 / 26777-7。我们感谢之前的研究(Franck等,2013; Quanico等,2015,2017a和2017b; Delcourt等,2017和2018),这些研究使我们能够执行和描述NAc显微解剖的方案。我们也感谢艺术家弗拉维亚Pagliusi (用于接触flaviapgl@gmail.com)是由遗传资源aphical方案适用于F igures 4 ,5和10 。
 
利益争夺
 
我们声明没有利益冲突。
 
伦理
 
此处介绍的所有实验均得到坎皮纳斯大学生物研究所动物使用伦理委员会-CEUA / UNICAMP的批准(协议3849-1和4249-1)。
 
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引用:Pagliusi Jr., M., Brandão, A. F., Zanetti, G. G., Bonet, I. J., Sartori, C. R. and Vieira, A. S. (2020). Using the Parafilm-assisted Microdissection (PAM) Method to Sample Rodent Nucleus Accumbens. Bio-protocol 10(23): e3836. DOI: 10.21769/BioProtoc.3836.
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