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Immunolocalization of Proteins in Corals: the V-type H+-ATPase Proton Pump
珊瑚中蛋白质的免疫定位:V型H+-ATPase质子泵   

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Abstract

Here we describe the immunolocalization of a membrane-bound proton pump, the V-type H+-ATPase (VHA), in tissues and isolated cells of scleractinian corals. Immunolocalization of coral proteins requires additional steps not required for various model organisms, such as decalcification of the coral skeleton for immunohistochemistry or removal of cells away from the skeleton for immunocytochemistry. The tissue and cell preparation techniques described here can be adapted for localization of other coral proteins, provided the appropriate validation steps have been taken for the primary antibodies and species of coral used. These techniques are important for improving our understanding of coral cell physiology

Materials and Reagents

  1. Live coral
  2. Electron microscopy grade paraformaldehyde (16% solution in 10 ml ampules) (Electron Microscopy Sciences, catalog number: 15700 or 15710 )
  3. Ethyl alcohol (absolute, 200 proof)
  4. Ethyl alcohol (190 proof)
  5. Xylenes (any reagent ≥ 98.5% xylenes, other tissue clearing agents such as Safeclear, are also acceptable) (Safeclear, catalog number: 044-192 )
  6. Paraffin embedding medium (e.g. Paraplast X-TRA) (McCormick Scientific, catalog number: 39503002 )
  7. Triton-X-100 (Bio-Rad Laboratories, AbD Serotec®, catalog number: 1610407 or Sigma-Aldrich, catalog number: T8787 )
  8. Normal goat serum (Vector laboratories, catalog number: S-1000 )
  9. Keyhole Limpet Hemocyanin (Sigma-Aldrich, catalog number: 7017 )
  10. Phosphate buffered saline (PBS) (e.g. 10x) (Corning Incorporated, catalog number: 46-013-CM )
  11. Microscope slides and coverslips
  12. Custom, antigen-affinity purified, rabbit polyclonal anti-VHAB antibodies developed using a peptide antigen matching a conserved region of the VHAB subunit (AREEVPGRRGFPGY) (Genscript USA, Inc.). The epitope is 100% conserved among diverse animal species, and specifically recognizes VHAB from coral (Barott et al., 2015), bone-eating worms (Tresguerres et al., 2013), hagfish (Clifford, et al. 2015), and sharks (Tresguerres et al., 2010; Roa et al., 2014).
    Note: This protocol may be adapted for other proteins following validation of the primary antibodies in the coral species to be examined. However, successful localization using different primary antibodies may require optimization of the fixation procedure from that described below.
  13. Secondary antibodies: Goat anti-rabbit IgG Alexa Fluor 555 (2 mg/ml) (Life Technologies, catalog number: A21429 )
    Note: The Alexa555 fluorophore is used to avoid overlap with the coral’s endogenous green fluorescent protein (GFP), but other fluorophores outside of the GFP emission range may also work.
  14. Hoescht 33342 (Life Technologies, catalog number: H1399 )
  15. Mounting medium (e.g. Electron Microscopy Sciences, catalog number: 17895 )
    Note: Unless otherwise noted, reagents can be obtained from any general laboratory reagent manufacturer or supplier.
  16. S22 buffer (see Recipes)
  17. Ca-free S22 buffer + 0.5 M EDTA (see Recipes)
  18. Fixative solution (see Recipes)
  19. Blocking buffer (see Recipes)

Equipment

  1. Chemical fume hood
  2. Fluorescence microscope (e.g. Zeiss AxioObserver Z1, or any other microscope with the ability to detect blue (“DAPI filter”, peak excitation 350 nm; peak emission 461 nm; for Hoescht staining of nuclei) and red (“TRITC filter”, peak excitation 550 nm; peak emission 570 nm; for secondary antibodies) fluorescence.
  3. Optional
    1. Coral endogenous GFP and chlorophyll from dinoflagellate symbionts (Symbiodinum) are visible using settings for “green fluorescence” (“FITC filter”, peak excitation 490 nm; peak emission 525 nm).
    2. Structured illumination (Zeiss Apotome2), confocal capabilities *suggested but not required.
    3. Differential Interference Contrast (DIC) (Nomarski microscopy) *suggested but not required.
  4. Microcentrifuge [any model capable of spinning 1.5 ml tubes at 3,000 x g at 4 ℃
    Note: This temperature can be achieved by placing the microcentrifuge inside of a fridge.
  5. Tissue cassettes (e.g. C&A Scientific, catalog number: EC-0109x )
  6. Tissue embedding station or any other method to pour liquid paraffin (e.g. Kedee, model: KD-BM II )
  7. Embedding base molds (metal) (e.g. C&A Scientific, catalog number: HB-07 , 15 , 24 , 30 )
  8. Embedding rings (plastic) (e.g. Biologix, catalog number: 41-3004 )
  9. Microtome (any model capable of cutting 5-30 μm paraffin sections)
  10. Slide warmer/hot plate (any model capable of warming up slides to 30 ℃)
  11. Hydrobarrier pen (Pap pen) (e.g. Vector Laboratories, catalog number: H-4000 )
  12. Rotating platform (any rotator/shaker/rocker)

Software

  1. Imaging software (e.g. Zeiss Axiovision software, Adobe Photoshop, ImageJ)

Procedure

  1. Tissue preparation
    Note: Section A takes approximately 10-14 days when completed without stopping. There are several stopping places within Section A.
    1. Immerse a live coral fragment (~1-2 cm in length) in fixative solution [S22 buffer with 3% paraformaldehyde (PFA), see Recipes] and incubate overnight at 4 °C (Puverel et al., 2005).
      Notes:
      1. PFA stock solution should be handled in a chemical fume hood.
      2. It is best to add PFA stock solution (16% or 32% PFA) to S22 buffer immediately before use. Diluted PFA can be stored at 4 °C for up to one week. Stock PFA may be stored at 4 °C for up to one month or at -20 °C for 6 months once unsealed.
    2. Decalcify by transferring fragment to Ca-free S22 buffer with 0.5 M EDTA and 0.5% paraformaldehyde and incubate at 4 °C. Replace the buffer daily until the skeleton is completely dissolved (~7 days depending on size of fragment and thickness/density of skeleton). Dissolution of the skeleton is determined visually; the coral tissue should appear transparent with no visible white calcium carbonate remaining (Figure 1).
      Note: It is best to add PFA stock solution (16% or 32% PFA) to Ca-free S22 buffer immediately before use.


    Figure 1. Example of a fully decalcified coral fragment. Only the tissue remains.

    1. Dehydrate tissue.
      Note: Incubations in steps a-f are conducted on a shaking platform.
      1. Incubate in 50% ethanol for 2-5 h.
      2. Incubate in 70% ethanol overnight at 4 °C.
        1. Tissue will keep in 70% ethanol at 4 °C for months.
      3. Put tissue in mesh plastic tissue cassette for final incubation steps in glass beaker.
      4. Incubate in 95% ethanol for 20 min.
      5. Incubate in 100% ethanol for 20 min. Repeat with fresh ethanol for a total of three times.
      6.  Incubate in xylene for 20 min. Repeat with fresh xylene for a total of three times.
        1. **Do in HOOD.
      7. Incubate in paraffin for 30 min. Repeat with fresh paraffin for a total of three times.
    2. Embed tissue in paraffin wax.
      1. Transfer tissue to base mold and position as desired.
        1. Fill with wax.
      2. Place embedding ring on top of base mold, and fill with wax to the top.
      3. Let wax solidify at room temperature for 1-2 days.
      4. Embedded tissue may be stored at room temperature* for several months prior to sectioning; long-term, archival storage must be done at 4 °C (*but storage at 4 °C is recommended).
    3. Sectioning.
      1. Turn on hot plate to 40 °C.
      2. Trim wax block around the coral tissue into the shape of a trapezoid (i.e. so top and bottom are parallel, bottom edge longer, and left half square; Figure 2).


        Figure 2. Example of coral tissue embedded in paraffin wax before (top) and after trimmed (bottom) in preparation for sectioning by a microtome

      3. Load sample onto microtome.
      4. Load blade with holder.
        1. Blade stored at -20 °C.
        2. Blade set at 15 degree angle (may be adjusted).
      5. Unlock stage and slide until blade almost touching sample.
      6. Start cutting sample using thick (15-30 μm) sections until surface is flat and smooth and tissue is reached.
      7. Switch to desired tissue thickness for slides (5-10 μm).
        1. Should get ribbon of consecutive sections (each section is easily identifiable because the right edge of each section is not parallel to the left edge of the following section).
      8. Cut ribbon into individual sections with razor blade and place onto glass microscope slides.
        1. Blade-cut side down.
        2. Label slides with pencil.
      9. Float section on water, remove any bubbles (two or three consecutive sections can be placed on each slide).
      10. Stretch wax section by placing slide on hot plate (~30 sec). Surface should become smooth.
        1. Remove water with Kim wipe.
      11. Let dry/affix to slide by leaving slide on hotplate overnight at 30 °C with paper towel underneath.
      12. Store at 4 °C until use.
    1. Deparaffinization and rehydration.
      1. Incubate in xylene bath for 10 min. Repeat with fresh xylene for a total of three times.
        1. **Do in HOOD.
      2. Incubate in 100% ethanol bath for 10 min.
      3. Incubate in 95% ethanol bath for 10 min.
      4. Incubate in 70% ethanol bath for 10 min.
      5. Incubate in PBS-TX (1x PBS with 0.2% (v/v) Triton-X-100) bath for 10 min.

    B. Isolated cell preparation
    1. Isolated coral cells were prepared from A. yongei colonies as described previously (Venn et al., 2009; Barott et al., 2015). Briefly, a coral fragment was submerged in 0.2 μm filtered seawater and the tissue was removed from the skeleton by gentle brushing with a toothbrush. Cells were then pelleted by centrifugation (3,000 x g for 4 min at 4 °C) and resuspended in the desired solution (see step B2 below).
    2. Fix cells by incubating in S22 buffer with 4% paraformaldehyde for 15 min on ice.
    3. Pellet cells (3,000 x g for 4 min at 4 °C) to remove the fixative. Resuspend and concentrate cells in 500 µl S22 buffer.
    4. Spread onto glass slides and air-dry. Optional: This step may be repeated several times to increase the cell concentration on each slide.
    5. Stain sample right away as described in section C below; if absolutely necessary slides may be stored at 4 °C for up to 1 h prior to staining.

    C. Staining
    1. Draw circle around tissue with hydrobarrier pen (Figure 3).


      Figure 3. Example of slide containing three tissue sections encircled by hydrobarrier

    2. Permeabilize tissue sections or isolated cells by adding PBS-TX to the top of the section/cells (~70-100 µl).
      1. Incubate for 60 or 10 min, respectively.
    3. Remove liquid using pipet. Incubate samples with 70-100 µl blocking buffer for 1 h at room temperature.
    4. Incubate samples with the anti-VHAB antibodies (3 µg/ml in blocking buffer) overnight at 4 °C in a humidified chamber.
    5. For secondary antibody controls, blocking buffer with no primary antibody is used on a section on the same slide.
      Note: Antibody specificity must be confirmed for each new species and antibodies pool; see below.
    6. Rinse in sequential baths of PBS-TX for 5 min each (3 times total).
    7. Incubate with the secondary antibody (goat anti-rabbit-Alexa 555; 1:500 dilution in blocking buffer) for 1 h at room temperature (secondary antibodies with other fluorophores may also be used; however, many coral cells, especially those in the polyp tentacles, contain very bright green fluorescent green protein that masks fluorophores such as Alexa 488 and similar).
    8. Incubate with Hoescht (1 µg/ml) for 5 min at room temperature to stain DNA.
      1. Rinse in sequential baths of PBS-TX for 5 min each (3 times total).
    9. Add ~50 µl mounting gel to top of each section.
      1. Place glass cover slip on top.
      2. Smooth away bubbles from tissue region by sliding the coverslip around (do not pick up cover as it may damage the tissue).
    10. Dab edges of cover slip with nail polish to secure, allow to dry.
    11. Store at 4 °C until imaging.

    D. Validation of anti-VHAB antibodies specificity
    Note: Validation of antibodies by Western blot should be done on homogenized samples. A single, clean band of approximately 55 KDa should be obtained for anti-VHAB antibodies, this band should not be present in peptide pre-absorption control, pre-immune serum control, or when the primary antibodies are omitted. Roa et al. (2014) describes peptide pre-absorption controls in detail, and Barott et al. (2015) describes preparation of coral samples for polyacrylamide gel electrophoresis and Western blot. These same controls should be carried out for IHC, and should lead to low background signal (see example in Figure 4).
    1. Prepare the following solutions in blocking buffer:
      1. Pre-immune serum control (3 µg/ml).
      2. Peptide pre-absorption control (3 µg/ml anti-VHAB antibodies plus 6 µg/ml peptide antigen). Incubate in rotating platform overnight at 4 °C.
      3. Anti-VHAB antibodies (3 µg/ml). Incubate in rotating platform overnight at 4 °C.
    2. Incubate samples as described in “Procedure C”. Staining, steps C4-10.

    E. Microscopy
    1. Observe and capture images using standard immunofluorescence microscopy protocols (epifluorescence, structured illumination, confocal), and appropriate excitation/emission settings for each fluorophore.
    2. If using Alexa555 secondary antibodies, VHA immunofluorescence is visible in the red channel (Figure 4). At longer exposure times, chlorophyll autofluorescence from dinoflagellate symbionts (Symbiodinum), will also be visible.
    3. Adjust the settings to the longest exposure time that results in maximum VHA signal from host coral cells and minimum or no chlorophyll signal. Control sections should show no red fluorescence under using the same settings.
    4. Visualize nuclei using blue fluorescence channel settings. Nuclei in coral host cells appear as solid circles, while nuclei in symbionts generally display a punctate staining pattern (possibly caused by super-coiled chromosomes).
    5. Optional 1: Visualize coral endogenous green fluorescence proteins and symbiont chlorophyll autofluorescence using green fluorescence channel settings.
    6. Optional 2: Visualize general tissue morphology using DIC.
    7. Use imaging software to adjust digitalized images for brightness and contrast only over the entire image (and following standard image manipulation guidelines).

Representative data



Figure 4. Immunofluorescence localization of VHA in tissues of the coral Acropora yongei. A. Control (primary antibodies omitted), B. anti-VHAB antibodies (red). Hoescht staining of nuclei is shown in blue.

Recipes

  1. S22 buffer
    1. 450 mM NaCl
    2. 10 mM KCl
    3. 58 mM MgCl2
    4. 10 mM CaCl2
    5. 100 mM Hepes (pH 7.8)
      Note: Adjust pH of Hepes buffer with NaOH.
  2. Ca-free S22 buffer + 0.5 M EDTA
    1. a450 mM NaCl
    2. 10 mM KCl
    3. 58 mM MgCl2
    4. 0.5 M EDTA
    5. 100 mM Hepes (pH 7.8)
      Note: Adjust pH of Hepes buffer with NaOH.
  3. Fixative solution (10 ml)
    1. Break open PFA ampule (16% PFA stock solution).
    2. Add 1.9 ml PFA stock solution to 8.1 ml S22 buffer (3% PFA final concentration). Invert to mix.
      Notes:
      1. Remaining PFA stock solution can be stored at 4 °C for up to one month or at -20 °C for 6 months.
      2. Fixative solution can be stored at 4 °C for up to one week.
  4. Blocking buffer
    1. 4 ml PBS-0.2% TritonX
    2. 80 µl NGS (normal goat serum)
    3. 0.8 µl KLH (Keyhole Limpet Hemocyanin) solution

Acknowledgments

This work was supported by National Science Foundation Grants EF-1220641 (to M. T.) and OCE-1226396 (to K. L. B.), and by Alfred P. Sloan Research Fellowship Grant BR2013-103 (to M. T.). We thank Sidney Perez for assistance with the isolated cell staining protocol.

References

  1. Barott, K. L., Venn, A. A., Perez, S. O., Tambutte, S. and Tresguerres, M. (2015). Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis. Proc Natl Acad Sci U S A 112(2): 607-612.
  2. Clifford, A., Goss, G. G., Roa, J. N. and Tresguerres, M. (2015). in Hagfish Biology [Science Publishers, CRC Press, Boca Raton, FL (In press)].
  3. Puverel, S., Tambutté, E., Zoccola, D., Domart-Coulon, I., Bouchot, A., Lotto, S., Allemand, D. and Tambutté, S. (2005). Antibodies against the organic matrix in scleractinians: a new tool to study coral biomineralization. Coral Reefs 24(1): 149-156.
  4. Roa, J. N., Munevar, C. L. and Tresguerres, M. (2014). Feeding induces translocation of vacuolar proton ATPase and pendrin to the membrane of leopard shark (Triakis semifasciata) mitochondrion-rich gill cells. Comp Biochem Physiol A Mol Integr Physiol 174: 29-37.
  5. Tresguerres, M., Katz, S. and Rouse, G. W. (2013). How to get into bones: proton pump and carbonic anhydrase in Osedax boneworms. Proc Biol Sci 280(1761): 20130625.
  6. Tresguerres, M., Parks, S. K., Salazar, E., Levin, L. R., Goss, G. G. and Buck, J. (2010). Bicarbonate-sensing soluble adenylyl cyclase is an essential sensor for acid/base homeostasis. Proc Natl Acad Sci U S A 107(1): 442-447.
  7. Venn, A. A., Tambutte, E., Lotto, S., Zoccola, D., Allemand, D. and Tambutte, S. (2009). Imaging intracellular pH in a reef coral and symbiotic anemone. Proc Natl Acad Sci U S A 106(39): 16574-16579.

简介

在这里我们描述膜结合质子泵,V型H + -ATPase(VHA),在组织和分离的细胞的scleractinian珊瑚的免疫定位。 珊瑚蛋白的免疫定位需要对于各种模型生物不需要的额外步骤,例如用于免疫组织化学的珊瑚骨架的脱钙或从用于免疫细胞化学的骨架移除细胞。 本文所述的组织和细胞制备技术可适用于其它珊瑚蛋白的定位,条件是已对所用的一级抗体和珊瑚种类采取了适当的验证步骤。 这些技术对于提高我们对珊瑚细胞生理学的理解是重要的

材料和试剂

  1. 活珊瑚
  2. 电子显微镜级多聚甲醛(10%安瓿中的16%溶液)(Electron Microscopy Sciences,目录号:15700或15710)
  3. 乙醇(绝对,200标准)
  4. 乙醇(190证明)
  5. 二甲苯(任何试剂≥98.5%二甲苯,其他组织清除剂如Safeclear,也是可接受的)(Safeclear,目录号:044-192)
  6. 石蜡包埋介质(例如 Paraplast X-TRA)(McCormick Scientific,目录号:39503002)
  7. Triton-X-100(Bio-Rad Laboratories,AbD Serotec ,目录号:1610407或Sigma-Aldrich,目录号:T8787)
  8. 正常山羊血清(Vector laboratories,目录号:S-1000)
  9. 匙孔血蓝蛋白(Sigma-Aldrich,目录号:7017)
  10. 磷酸盐缓冲盐水(PBS)(例如10x)(Corning Incorporated,目录号:46-013-CM)
  11. 显微镜载玻片和盖玻片
  12. 使用与VHA亚基亚基(AREEVPGRRGFPGY)(Genscript USA,Inc。)的保守区匹配的肽抗原开发的定制的抗原亲和纯化的兔多克隆抗VHA B抗体。)。所述表位在不同动物物种中是100%保守的,并且特异性识别来自珊瑚的VHA亚基(Barott等人,2015),骨摄食蠕虫(Tresguerres) et al。,2013),hagfish(Clifford, 2015)和鲨鱼(Tresguerres等人,2010; Roa等人al。,2014)。
    注意:此协议可以适应其他蛋白质,在验证的一级抗体在珊瑚物种中检查。然而,使用不同的一抗的成功定位可能需要从下面描述的固定程序的优化。
  13. 二抗:山羊抗兔IgG Alexa Fluor 555(2mg/ml)(Life Technologies,目录号:A21429)
    注意:Alexa555荧光团用于避免与珊瑚的内源性绿色荧光蛋白(GFP)重叠,但GFP发射范围之外的其他荧光团也可能起作用。
  14. Hoescht 33342(Life Technologies,目录号:H1399)
  15. 安装介质(例如,Electron Microscopy Sciences,目录号:17895)
    注:除非另有说明,试剂可从任何一般实验室试剂制造商或供应商处获得。
  16. S22缓冲区(参见配方)
  17. 无钙S22缓冲液+ 0.5M EDTA(参见配方)
  18. 固定解决方案(参见配方)
  19. 阻塞缓冲区(参见配方)

设备

  1. 化学通风橱
  2. 荧光显微镜(例如Zeiss AxioObserver Z1或具有检测蓝色的能力的任何其他显微镜("DAPI滤光片",峰值激发350nm;峰值发射461nm;对于核的Hoescht染色)和红色 "TRITC过滤器",峰值激发550nm;峰值发射570nm;对于二次抗体)荧光
  3. 可选
    1. 珊瑚内源性GFP和叶绿素从鞭毛藻共生体 ( )可以使用"绿色荧光"("FITC   滤波器",峰值激发490nm; 峰值发射525nm)
    2. 结构照明(Zeiss Apotome2),共焦能力*建议,但不是必需的。
    3. 差分干涉对比(DIC)(Nomarski显微镜)*建议,但不是必需的。
  4. 微量离心机[任何能够在4℃以3000×g/min旋转1.5ml试管的型号]
    注意:这个温度可以通过将微量离心机放在冰箱里实现。
  5. 组织盒(如 C& A Scientific,目录号:EC-0109x)
  6. 组织包埋站或任何其他方法倒入液体石蜡(例如Kedee,型号:KD-BM II)
  7. 嵌入基础模具(金属)(如 C& A Scientific,目录号:HB-07,15,24,30)
  8. 嵌入戒指(塑料)(例如 Biologix,目录号:41-3004)
  9. 切片机(任何能切割5-30μm石蜡切片的型号)
  10. 幻灯片加热器/热板(任何能够将幻灯片预热到30℃的型号)
  11. Hydrobarrier pen(Pap pen)(例如Vector Laboratories,目录号:H-4000)
  12. 旋转平台(任何旋转器/振动器/摇杆)

软件

  1. 成像软件(例如Zeiss Axiovision软件,Adobe Photoshop,ImageJ)

程序

  1. 组织准备
    注意:完成后不需停止,A节大约需要10-14天。 A区内有几个停车位。
    1. 沉浸在固定剂中的活珊瑚碎片(长度约1-2厘米) 溶液[具有3%多聚甲醛(PFA)的S22缓冲液,参见配方]和 在4℃下孵育过夜(Puverel等人,2005)。
      注意:
      1. PFA储备溶液应在化学通风橱中处理。
      2. 最好向S22缓冲液中加入PFA储备溶液(16%或32%PFA) 立即使用。 稀释的PFA可以在4℃下储存多达一个 周。 储备PFA可以在4℃下储存长达一个月或-20℃ 已开封6个月。
    2. 通过传递碎片进行脱钙   无钙S22缓冲液,含0.5M EDTA和0.5%多聚甲醛 在4℃孵育。 每天更换缓冲区,直到骨架 完全溶解(约7天,取决于片段的大小) 厚度/骨架密度)。 骨骼的溶解是 视觉确定; 珊瑚组织应该看起来透明,没有 可见白色碳酸钙剩余(图1) 注意:最好在使用前立即向无Ca的S22缓冲液中加入PFA储备溶液(16%或32%PFA)。


    图1.完全脱钙的珊瑚碎片的示例。 只剩下组织。

    1. 脱水组织。
      注意:步骤a-f中的孵化在振动平台上进行。
      1. 在50%乙醇中孵育2-5小时
      2. 在70%乙醇中于4℃孵育过夜
        1. 组织将保持在70%乙醇在4°C数个月。
      3. 将组织放入网状塑料组织盒中,在玻璃烧杯中进行最后的温育步骤
      4. 在95%乙醇中孵育20分钟
      5. 在100%乙醇中孵育20分钟。 用新鲜乙醇重复三次。
      6.  在二甲苯中孵育20分钟。 用新鲜二甲苯重复三次。
        1. **在HOOD。
      7. 在石蜡中孵育30分钟。 用新鲜石蜡重复总共三次。
    2. 将组织嵌入石蜡中。
      1. 根据需要将纸巾转移到基础模具和位置。
        1. 填充蜡。
      2. 将嵌入环放置在基础模具的顶部,并向顶部填充蜡。
      3. 让蜡在室温下固化1-2天
      4. 包埋的组织可以在室温下储存几个 切片前几个月; 长期,档案存储必须在4   °C(*,建议储存在4°C)。
    3. 切片。
      1. 将加热板打开至40°C。
      2. 修剪蜡块围绕珊瑚组织成梯形   (即,因此顶部和底部是平行的,底部边缘更长,左半部分   广场; 图2)。


        图 2.嵌入在石蜡中的珊瑚组织的实例(顶部)和 修剪(底部)以准备切片机切片

      3. 将样品装载到切片机上。
      4. 用刀柄装载刀片。
        1. 刀片储存于-20°C
        2. 刀片设置在15度角(可以调整)。
      5. 解锁台架并滑动,直到刀片几乎接触到样品
      6. 使用厚(15-30μm)切片开始切割样品,直到表面平整光滑,达到组织
      7. 切换到幻灯片所需的组织厚度(5-10μm)。
        1. 应该得到连续部分的丝带(每个部分很容易 可识别,因为每个部分的右边缘不平行 下一节的左边缘)。
      8. 用刀片将带切成单独的切片,放在玻璃显微镜载玻片上
        1. 刀片侧向下。
        2. 用铅笔标签幻灯片。
      9. 浮在水面上,去除任何气泡(每个幻灯片上可以放置两个或三个连续的部分)
      10. 通过将载玻片放置在热板上(约30秒)来拉伸蜡部分。 表面应该光滑。
        1. 用Kim wipe清除水。
      11. 让干/贴在幻灯片上,在30℃下用电热板过夜,用纸巾在下面
      12. 储存于4°C直至使用。
    1. 脱蜡和再水合。
      1. 在二甲苯浴中孵育10分钟。 用新鲜二甲苯重复三次。
        1. **在HOOD。
      2. 在100%乙醇浴中孵育10分钟。
      3. 在95%乙醇浴中孵育10分钟
      4. 在70%乙醇浴中孵育10分钟
      5. 在PBS-TX(1x PBS与0.2%(v/v)Triton-X-100)浴中孵育10分钟。

    B.分离细胞制备
    1. 从A制备分离的珊瑚细胞。 yongei 殖民地 (Venn等人,2009; Barott等人,2015)。 简而言之,a   将珊瑚片段浸没在0.2μm过滤的海水和组织中   通过用牙刷轻轻刷牙从骨骼中除去。 然后通过离心(3,000×g,4℃,4℃)沉淀细胞,   并重新悬浮在所需的溶液中(见下面的步骤B2)
    2. 修复细胞通过在S22缓冲液中与4%多聚甲醛在冰上孵育15分钟。
    3. 沉淀细胞(3,000×g在4℃下4分钟)以除去固定剂。 重悬和浓缩细胞在500微升S22缓冲液。
    4. 传播到玻璃载片上并风干。 可选:此步骤可以 重复几次以增加每个载玻片上的细胞浓度。
    5. 如下面C节所述立即染色样品; 如果 绝对必要的载玻片可以在4℃下储存多达1小时   染色

    C.染色
    1. 用水压笔在组织周围绘制圆圈(图3)。


      图3.包含由水力障碍包围的三个组织切片的幻灯片示例

    2. 通过添加PBS-TX到部分/细胞的顶部(〜70-100μl)来透化组织切片或分离的细胞。
      1. 分别孵育60或10分钟。
    3. 使用移液器除去液体。 在室温下用70-100μl封闭缓冲液孵育样品1小时
    4. 将样品与抗VHA B抗体(在封闭缓冲液中3μg/ml)在4℃下在加湿室中孵育过夜。
    5. 对于第二抗体对照,在相同载玻片的切片上使用没有第一抗体的封闭缓冲液。
      注意:必须对每个新物种和抗体库确认抗体特异性; 见下文。
    6. 在PBS-TX的连续浴中冲洗5分钟(共3次)
    7. 与第二抗体(山羊抗兔Alexa 555; 在封闭缓冲液中1:500稀释)在室温下1小时 (也可以使用具有其他荧光团的二抗;然而,   许多珊瑚细胞,特别是那些在息肉触手,包含非常   亮绿色荧光绿色蛋白,掩盖荧光团如 Alexa 488和类似)。
    8. 与Hoescht(1μg/ml)孵育5分钟,在室温下染色DNA。
      1. 在PBS-TX的连续浴中冲洗5分钟(共3次)。
    9. 在每个部分的顶部加入〜50μl安装凝胶
      1. 将玻璃盖放在顶部。
      2. 通过滑动盖玻片(不要拿起盖子,因为它可能会损坏组织),平滑的气泡从组织区域。
    10. 盖子的边缘用指甲油擦拭固定,使其干燥。
    11. 存储在4°C,直到成像。

    D.抗VHA亚型抗体特异性的验证
    注意:通过Western印迹验证抗体应该在匀浆样品上进行。对于抗VHA B抗体应该获得大约55KDa的单一清洁条带,该条带不应当存在于肽预吸收对照,免疫前血清对照中,或者当第一抗体被省略。 Roa等人(2014)详细描述了肽的预吸收控制,Barott et al。 (2015)描述了用于聚丙烯酰胺凝胶电泳和Western印迹的珊瑚样品的制备。这些相同的控制应该针对IHC进行,并且应当导致低背景信号(参见图4中的实例)。
    1. 在阻塞缓冲区中准备以下解决方案:
      1. 免疫前血清对照(3μg/ml)
      2. 肽预吸收对照(3μg/ml抗VHA B抗体加上6μg/ μg/ml肽抗原)。 在4℃下在旋转平台中孵育过夜。
      3. 抗VHA B抗体(3μg/ml)。 在4℃下在旋转平台中孵育过夜。
    2. 如"程序C"中所述孵育样品。 染色,步骤C4-10。

    E.显微镜
    1. 使用标准免疫荧光观察和捕获图像 显微镜方案(落射荧光,结构化照明, 共焦)和适当的激发/发射设置 荧光团
    2. 如果使用Alexa555二级抗体,VHA 免疫荧光在红色通道中可见(图4)。 更长 暴露时间,从鞭毛藻的叶绿素自体荧光 共生体( Symbiodinum )也将是可见的。
    3. 调整 设置为导致最大VHA信号的最长曝光时间   从主机珊瑚细胞和最小或没有叶绿素信号。 控制 部分在使用相同设置时不应显示红色荧光
    4. 使用蓝色荧光通道设置可视化核。 核   珊瑚宿主细胞呈现为实心圆,而核在共生体中 一般显示点状染色模式(可能由 超螺旋染色体)。
    5. 可选1:可视化珊瑚 内源性绿色荧光蛋白和共生叶绿素 使用绿色荧光通道设置的自发荧光。
    6. 可选2:使用DIC显现一般组织形态。
    7. 使用成像软件调整数字化图像的亮度和   仅在整个图像(和以下标准图像)上对比 操作指南)。

代表数据



图4. VHA在珊瑚藻类的组织中的免疫荧光定位。 A。 对照(省略了一级抗体),B.抗VHA B抗体(红色)。 Hoescht染色的核显示为蓝色。

食谱

  1. S22缓冲区
    1. 450 mM NaCl
    2. 10 mM KCl
    3. 58mM MgCl 2·h/v
    4. 10mM CaCl 2
    5. 100mM Hepes(pH7.8)
      注意:用NaOH调节Hepes缓冲液的pH。
  2. 无钙S22缓冲液+ 0.5M EDTA
    1. a450mM NaCl
    2. 10 mM KCl
    3. 58mM MgCl 2·h/v
    4. 0.5 M EDTA
    5. 100mM Hepes(pH7.8)
      注意:用NaOH调节Hepes缓冲液的pH。
  3. 固定溶液(10ml)
    1. 打开PFA安瓿(16%PFA储备液)。
    2. 将1.9ml PFA储备溶液加入到8.1ml S22缓冲液(3%PFA终浓度)中。 反转混合。
      注意:
      1. 剩余的PFA储备液可以在4℃下储存长达一个月或在-20℃下储存6个月。
      2. 固定溶液可以在4℃下储存长达一周。
  4. 阻塞缓冲区
    1. 4ml PBS-0.2%TritonX /
    2. 80μlNGS(正常山羊血清)
    3. 0.8μlKLH(匙孔血蓝蛋白)溶液

致谢

这项工作由国家科学基金会资助EF-1220641(对M.T.)和OCE-1226396(对K.L.B)和Alfred P. Sloan研究奖学金资助BR2013-103(对M.T.)支持。 我们感谢Sidney Perez对分离细胞染色方案的帮助。

参考文献

  1. Barott,K.L.,Venn,A.A.,Perez,S.O.,Tambutte,S。和Tresguerres,M。(2015)。 珊瑚宿主细胞酸化共生藻类微环境以促进光合作用。 Proc Natl Acad Sci USA 112(2):607-612。
  2. Clifford,A.,Goss,G.G.,Roa,J.N.和Tresguerres,M。(2015)。 in Hagfish Biology [Science Publishers,CRC Press,Boca Raton,FL(In press)]。
  3. Puverel,S.,Tambutté,E.,Zoccola,D.,Domart-Coulon,I.,Bouchot,A.,Lotto,S.,Allemand,D.andTambutté, 抗角化细胞有机基质的抗体:一种研究珊瑚生物矿化的新工具。/a> 珊瑚礁 24(1):149-156。
  4. Roa,J. N.,Munevar,C.L.和Tresguerres,M.(2014)。 喂养诱导液泡质子ATP酶和裙蛋白移位到豹鲨的膜上( Triakis semifasciata )线粒体富集的细胞。
  5. Tresguerres,M.,Katz,S。和Rouse,G.W。(2013)。 如何进入骨骼: Osedax boneworms中的质子泵和碳酸酐酶 。 Proc Biol Sci 280(1761):20130625.
  6. Tresguerres,M.,Parks,S.K.,Salazar,E.,Levin,L.R.,Goss,G.G。和Buck,J。(2010)。 碳酸氢盐感应可溶性腺苷酸环化酶是酸/碱基稳态的重要传感器。 Proc Natl Acad Sci USA 107(1):442-447。
  7. Venn,A.A.,Tambutte,E.,Lotto,S.,Zoccola,D.,Allemand,D。和Tambutte,S。(2009)。 在珊瑚礁和共生海葵中成像细胞内pH。美国国家科学院Sci USA 106(39):16574-16579。
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引用:Barott, K. L. and Tresguerres, M. (2015). Immunolocalization of Proteins in Corals: the V-type H+-ATPase Proton Pump. Bio-protocol 5(17): e1573. DOI: 10.21769/BioProtoc.1573.
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