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Immunogold Electron Microscopy of the Autophagosome Marker LC3
自噬体标志物LC3的免疫金电镜观察   

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PLOS Pathogens
Mar 2017

Abstract

Even though autophagy was firstly observed by transmission electron microscopy already in the 1950s (reviewed in Eskelinen et al., 2011), nowadays this technique remains one of the most powerful systems to monitor autophagic processes. The autophagosome, an LC3-positive double membrane structures enclosing cellular materials, represents the key organelle in autophagy and its simple visualization and/or numeration allow to draw important conclusions about the autophagic flux. Therefore, the accurate identification of autophagosomes is crucial for a comprehensive and detailed dissection of autophagy. Here we present a simple protocol to identify autophagosomes by transmission electron microscopy coupled to immunogold labeling of LC3 starting from a relatively low cell number, which we recently developed to follow the autophagic pathway during viral-mediated human carcinogenesis.

Keywords: Autophagy (自噬), Immunogold electron microscopy (免疫金电微镜技术), Autophagosome (自噬小体), LC3 (LC3), Viral carcinogenesis (病毒致癌作用)

Background

The autophagosome represents the key structure in macroautophagy, a catabolic degradation system for cellular cytosolic constituents. Macroautophagy (or simply autophagy) is initiated by the formation of the phagophore, a membrane able to expand itself engulfing organelles and proteins that finally closes around sequestered components forming an organelle known as autophagosome. Next, during the maturation process, the autophagosome can fuse with lysosomes to form autolysosomes where trapped materials are degraded and recycled back by pumps located in the lysosomal limiting membrane (Glick et al., 2010).

Since malfunction of autophagy has been linked to a variety of human disease, viral infection, neurodegeneration, immune function, and cancer (Schneider and Cuervo, 2014), a more comprehensive and detailed dissection of autophagy is increasingly required. Indeed, numerous methodologies to study autophagy have been developed so far (Klionsky et al., 2016), and most of them need to be simultaneously performed in order to correctly define how autophagy is working in a particular experimental system. Among them, transmission electron microscopy (TEM) coupled with specific immunogold labeling (immunogold electron microscopy-IEM) remains one of the most accurate methods for autophagy detection because of its exquisite ability to provide fine details of specific ultracellular structures. Indeed, in addition to their peculiar morphology (Yla-Anttila et al., 2009), autophagosomes are also characterized by the presence of microtubule-associated protein 1 light chain 3, or LC3, which is generally recruited on autophagic membranes after lipidation with phosphatidylethanolamine (LC3-II) during autophagic activation (Kabeya et al., 2000). Therefore, TEM coupled with labeling of cells with anti-LC3 antibody and gold probes conjugated to a secondary antibody allows the unequivocal identification of the autophagosome, and indirectly, permits to monitor autophagic flux.

Several approaches are commonly used for the immunogold labelling of cultured cells: pre-embedding (immunolabeling before resin embedding) and post-embedding methods (immunolabeling after resin embedding) including also Tokuyasu cryosections, where cryoprotected embedded specimens are firstly frozen by immersion in liquid nitrogen and then cut with cryo-ultramicrotome at -120 °C before immunolabelling (Tokuyasu, 1980; Eskelinen et al., 2002; Jager et al., 2004). However, both techniques require at least 3 x 106 cells to obtain an adequate pellet for sample preparation, inferring that initial cell number is a critical factor to consider during planning of IEM experiments.

Therefore, we describe herein a pre-embedding protocol for autophagosome detection starting from small cell numbers (less than 0.1 x 106 cells), which allows performing IEM analysis in primary cells characterized by low replicative potential. We recently used this protocol to detect LC3-positive autophagosomes on populations of primary human keratinocytes transduced with retroviral vectors relevant to viral carcinogenesis (Mattoscio et al., 2017). In principle, this methodology could be applied to detect other antigens of interest, since we used the same protocol also to study the subcellular localization of the SUMO-conjugating enzyme UBC9 (Mattoscio et al., 2017), and may be used with different cellular species for which the initial cell number is also a limiting factor.

Materials and Reagents

  1. 13 mm Nunc Thermanox coverslips (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150067 )
  2. 24-well tissue culture-treated plates (Corning, Costar®, catalog number: 3524 )
  3. Eppendorf® Safe-Lock microcentrifuge tubes (Sigma-Aldrich, catalog number: T9661)
    Manufacturer: Eppendorf, catalog number: 022363204 .
  4. Edge razor blade (Sigma-Aldrich, catalog number: Z740504 )
  5. 200 mesh grids (Sigma-Aldrich, catalog number: G5151 )
  6. Chang Monolayer Molds (Electron Microscopy Sciences, catalog number: 70920 )
  7. Ethanol BioUltra, for molecular biology (Sigma-Aldrich, catalog number: 51976 )
  8. Dulbecco’s phosphate-buffered saline, calcium, magnesium (Sigma-Aldrich, catalog number: D8662 )
  9. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
    Note: To prepare 4% paraformaldehyde (PFA) solution, in a fume hood dissolve 4 g of PFA in 90 ml of water at 60 °C and few drops of 1 N NaOH (until the solution appears without PFA precipitate), then remove from heating, add 10 ml 10x PBS, fill up with water up to 100 ml, and cool. Do not heat solution above 70 °C since PFA will break down at higher temperatures. Use the solution immediately or store for 1 week at 4 °C or at -20 °C for a longer time.
  10. Glutaraldehyde (Sigma-Aldrich, catalog number: G7776 )
  11. Quenching solution: 50 mM glycine (Sigma-Aldrich, catalog number: 410225 ) in PBS
  12. Primary antibody: rabbit polyclonal anti-LC3 (Novus Biologicals, catalog number: NB100-2331 )
  13. Washing buffer (0.1% BSA, 0.1% saponin in PBS)
  14. Secondary antibody conjugated with gold: 1.4 nm Nanogold IgG goat anti rabbit IgG (Nanoprobes, catalog number: 2003 )
  15. Gold enhancement solution (GoldEnhanceTM EM) (Nanoprobes, catalog number: 2113 ), made by four different components: A (enhancer), B (activator), C (initiator) and D (buffer)
  16. Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400 )
    Note: Uranyl acetate is mildly reactive and highly toxic by ingestion. Please dispose of waste according to your institutional policy.
  17. Embed-812 resin kit (Electron Microscopy Sciences, catalog number: 14120 )
    Notes:
    1. This product has been discontinued and replaced by EMbed 812.
    2. For a better preservation, mix Embed-812 (32 g) with Dodecenyl succinie anhydride (DDSA) (16 g) and NMA (Nadic Methyl Anhydride) (17.4 g). Mix well, add 0.8 g of the accelerator DMP30 (2,4,6-tri (dimethylaminomethyl) phenol), mix and store in 20 ml syringe at -20 °C. Warm to room temperature before use.
  18. Hydrofluoric acid (Fisher Scientific, catalog number: 14-650-236)
    Manufacturer: Avantor Performance Materials, J.T. Baker, catalog number: 9560-01 .
  19. Blocking buffer (see Recipes)
    1. Bovine serum albumin (BSA, IgG-Free and Protease-Free) (Jackson ImmunoResearch, catalog number: 001-000-162 )
    2. Normal goat serum (Jackson ImmunoResearch, catalog number: 005-000-121 )
    3. Ammonium chloride (Sigma-Aldrich, catalog number: A9434 )
    4. Saponin (Sigma-Aldrich, catalog number: 47036 )
    5. Phosphate buffer (Sigma-Aldrich, catalog number: P3619 )
    6. Sodium chloride (Sigma-Aldrich, catalog number: S7653 )
  20. Reduced osmium tetroxide (see Recipes)
    1. Osmium tetroxide (OsO4) (Electron Microscopy Science, catalog number: 19140 )
    2. Potassium ferrocyanide (Electron Microscopy Science, catalog number: 20150 )
    3. Cacodylate buffer pH 7.4 (from sodium cacodylate trihydrate) (Sigma-Aldrich, catalog number: C4945 )
  21. Sato lead solution (see Recipes)
    1. Lead citrate tribasic (Sigma-Aldrich, catalog number: 15326 )
    2. Lead nitrate (Merck, catalog number: 1073980100 )
    3. Sodium citrate tribasic dehydrate (Sigma-Aldrich, catalog number: S4641 )

Equipment

  1. Fume hood
  2. 60 °C Oven (Memmert, model: UN30 )
  3. Ultramicrotome (Leica Microsystem, model: Leica EM UC7 )
  4. Transmission Electron microscope (Carl Zeiss, model: Leo 912AB ) equipped with a slow-scan Proscan camera (ProScan)
  5. Rocking shaker

Procedure

  1. Fixation
    1. Sterilize the coverslips with ethanol, wash three times with PBS, and accommodate one slide for each well of the 24-well plate.
    2. Plate the cells to be tested on these slides in appropriate medium to obtain a 60-70% confluency on the following day.
      Note: To obtain enough technical replicates, we usually prepare 6 slides for each condition to be tested. In our study, we used primary keratinocytes expressing empty-control vector or a plasmid for the overexpression of the HPV oncoviral proteins E6/E7 (seeded about 1 x 105 cells/well) (Mattoscio et al., 2017). In addition, to unequivocally identify autophagosomes, appropriate positive and negative controls of autophagosome formation should be prepared. For example, consider activating/inhibiting autophagy with rapamycin 10-500 nM (to increase autophagosomes formation) or with class III PI3K inhibitors (such as 1-10 mM 3-methyladenine or 10-100 nM wortmannin to inhibit autophagosomes accumulation), respectively (Mizushima et al., 2010).
    3. The next day, remove the medium, wash 2 times with PBS, and fix cells in 4% freshly-prepared PFA in 0.1 M phosphate buffer pH 7.4 for 1 h at room temperature.
      Note: Since glutaraldehyde lowers protein antigenicity, although it provides best structural preservation, we prefer to initially fix cells with PFA alone, and then re-fix with glutaraldehyde after antigen labeling.
    4. Remove PFA, wash 2 times with PBS. Store fixed cells at 4 °C.
      Note: Since PFA fixation is reversible, for long time storage keep cells in storage solution (1% PFA in PBS) instead of PBS alone.

  2. Immunolabeling
    1. Remove fixative or storage solution, wash wells three times for 5 min each in PBS, and treat with quenching solution for 10 min at room temperature to quench the free aldehydes of the primary fixative.
    2. Permeabilize cells by incubation with 0.25% saponin, 0.1% BSA in PBS for 10 min at room temperature.
    3. Incubate cells for 30 min at room temperature with blocking buffer (0.2% BSA, 5% normal goat serum, 50 mM NH4Cl, 0.1% saponin, 20 mM phosphate buffer, 150 mM NaCl, see Recipes) to block non-specific staining.
    4. Remove blocking buffer and incubate coverslips with anti-LC3 primary antibody (1:150 dilution) in blocking buffer, for 2 h at room temperature.
    5. Wash cells with washing buffer (0.1% BSA, 0.1% saponin in PBS) 4 times for 5 min each.
    6. Incubate coverslips with 1.4 nm Nanogold goat anti rabbit secondary antibody conjugated with nanogold (1:200 dilution) in blocking buffer for 1 h at RT.
    7. Wash slides 3 times with washing buffer and wash three times in PBS.
    8. Re-fix stained cells with 1% glutaraldehyde in PBS for 30 min at room temperature to preserve ultrastructure. Rinse in PBS and leave in PBS at 4 °C until next step.

  3. Gold enhancement
    1. Equilibrate all the components of gold enhancement solution (A, B, C, D) at room temperature before use.
    2. Wash 3 x 5 min with 50 mM glycine in PBS.
    3. Wash 3 x 5 min with 1% BSA, 0.05% Tween in PBS.
    4. Rinse 3 times in PBS and in the meanwhile activate gold enhancement solution by adding in an Eppendorf tube one part each of component (A-enhancer and B-activator), mix well and incubate for 5 min.
      Note: Gold enhancement solution should be prepared immediately before use. Increasing the amount of component B might help in reducing nonspecific signals.
    5. Add to the gold enhancement mix one part of components C (initiator) and D (buffer), respectively, rinse cells in distilled water and add few drops of the solution to the coverslip. Prepare about 40 µl of gold enhancement solution per coverslip.
    6. Check the appearance of a dark purple/black precipitate (Figure 1). Incubation time with the gold enhancement solution may vary. Usually, it takes between 1 to 3 min for the full development. Always compare with the negative control (without primary antibody).
      Note: The goal of this step is to enlarge gold particles associated to the secondary antibody. To improve penetration and density labeling, gold particles of small size are preferable (1.4 nm). Since these nanogold particles will be hardly visible in samples, this step is mandatory. However, as an alternative to gold, a silver enhancement solution could be used. Silver enhancement is both commercially available (Nanoprobes, catalog number 2013) or can be prepared from single components (Stierhof et al., 1991). Silver enhancement is light sensitive and can be damaged by osmium tetroxide, therefore gold enhancement solution is usually preferred for its simplicity of use.
    7. Wash three times for 5 min each with distilled water.


      Figure 1. Gold enhancement. Put a drop of gold enhancement solution on the coverslips and incubate until a dark precipitate appears (visible by eye). Longer incubations may cause darker signals. Check under a bright field microscope or a stereomicroscope for a fine control of gold development. Note that the dark precipitate will not appear in the negative control (CTRL: no primary antibody). Bottom: magnification of coverslips at Time = 0 and at the end of gold enhancement incubation (T = 3 min). Please note that the dark precipitate appeared only where the primary antibody is present.

  4. Contrast enhancement
    1. Post-fix samples with reduced osmium tetroxide (see Recipes) for 1 h at RT. Wash 3 times for 5 min in 0.1 M sodium cacodylate and wash three times in distilled water.
      Note: The use of reduced osmium fixation step preserves membrane lipids. The addition of potassium ferrocyanide reduces the osmium making it more reactive to membranes, improving their contrast.
    2. En bloc stain with 1% uranyl acetate water solution overnight at 4 °C.
      Note: Do not expose to light. Uranyl acetate improves image contrast due to the high affinity of uranyl and lead ions for proteins, nucleic acids, and hydroxyl groups in carbohydrates and RNA, respectively. It is important to use water instead of alcoholic solution to better preserve cellular structures.

  5. Embedding
    1. Dehydrate specimens in an ascending ethanol series 30%, 50%, 70%, 80%, 90%, 96% for 5 min in each of the ascending series. Finally, dehydrate 3 times in 100% ethanol for 10 min every time.
    2. Replace absolute ethanol with a 1:1 solution of ethanol-embedding medium, on a rocking shaker for 2 h at room temperature.
    3. Remove coverslips from the ethanol-resin mix, absorb the excess of resin with a paper tissue and place the coverslips on top of a drop of resin. After 1 h, move the coverslips to a new drop of resin (Figure 2A).


      Figure 2. Embedding. Move the coverslip on drop of resin and incubate for one hour (A). Place a drop of Epon on a Chang Mold and lay the coverslips (B).

    4. Fill the cavities of a Chang monolayer Mold with few drops of resin and lay the coverslips (make sure to clean the top side of the coverslip from the excess of resin) on top of the cavity (Figure 2B).
    5. Place in the oven at 45 °C overnight, then increase the temperature to 60 °C and bake for further 24 h.

  6. Sectioning
    1. To detach Epon from slide, take the Epon block out of the oven and let it cool down. Then, immerse block in liquid nitrogen for 30 sec and then in hot water. This will detach resin containing embedded cells from the coverslip where cells were initially grown.
      Note: Alternatively, put the coverslips in a plastic multiwell, cover with a solution of concentrated hydrofluoric acid (30%), and shake for 5-10 min until the glass is completely dissolved.
    2. Wash the samples several times with distilled water, soak the samples in 0.1 M cacodylate buffer for 30 min, then wash with distilled water and dry.
    3. With a single edge razor blade remove a small portion of the resin block and stick with a cyanoacrylate glue on top of an empty resin block (Figure 3).


      Figure 3. Sectioning. After glass coverslips have been removed, cut with a razor blade a small portion of the resin block and glue on top of an empty resin block (plasticine, blue material in this image). After 1-2 h, the sample can be placed in an ultramicrotome for sectioning.

    4. Cut ultrathin sections of 60-90 nm thick and collect sections onto 200 mesh grids with square or hexagonal openings.
    5. Stain grids with 1% uranyl acetate water solution for 5 min and then Sato’s lead solution (see Recipes) for 2 min.
    6. Observe under an electron microscope and take digital micrographs.
      Note: To visualize autophagosomes, specimens should be observed at a magnification of 4,000 (for an overview) to 10,000x (to finely observe ultrastructure) looking for double-membraned organelles positive for LC3 gold particles, and containing undigested cytoplasmic contents (Figure 4A).

Data analysis

In this protocol, we applied a pre-embedding technique (immunolabeling before samples were embedded in resin) to detect LC3 in low-abundant cells preparation. We recently used this method to image both UBC9 and LC3-positive autophagosomes in primary human keratinocytes expressing either an Empty vector or HPV E6/E7 oncoviral proteins (Mattoscio et al., 2017). Indeed, TEM analysis showed the presence of UBC9 inside double-membranes intracellular structures that we identified as autophagic compartments by LC3-immunogold TEM, suggesting an autophagic dependent route for UBC9 degradation. 
Although TEM is one of the best technique to evaluate the autophagic process, the fact that autophagy is a dynamic process makes it difficult to extrapolate meaningful data from a single ultrastructural snapshot. Moreover, some late autophagic structures are difficult to identify due to the degradative process that takes place upon fusion to lysosomes. In Figure 4 we reported the morphology of the autophagic structures (indicated by arrows) in human keratinocytes comparing side by side the classical EM preparation (left) with LC3 immunolabeling (right), illustrating typical results obtained following our strategy. The combination of the initial mild fixation with PFA alone and the pre-embedding staining allowed a better antigen preservation despite preserving detailed information on the cell structure (Figure 4) and the labeling of cells with anti-LC3 antibody allows the unequivocal identification of the autophagic vacuoles. Moreover, cell fixation directly on microscopy slides without the need to scrape and pellet cells allows to work with a relatively low cell number and to maintain cell ultrastructure. Therefore, we believe that this protocol could be easily applied to detect additional low-abundant antigens in rare cell populations. However, a limitation of this approach is that it does not enable colocalization studies, which can be achieved only with post-embedding EM using gold probes of different sizes.


Figure 4. Autophagosomes identification using TEM and LC3 immunostaining. A. Left: TEM image of primary human keratinocytes. Right: TEM coupled with LC3 immunogold labeling. Arrows indicate autophagic vacuoles. Note that gold particles (LC3) are selectively enriched in autophagic structures. B. Magnification of the autophagic vacuole circled in Figure 4A (right). Note the membrane structure, LC3 positivity, and fusion with a lysosome (darker body). 

Recipes

  1. Blocking buffer
    0.2% bovine serum albumin (BSA, IgG-Free and Protease-Free)
    5% normal goat serum
    50 mM ammonium chloride
    0.1% saponin
    20 mM phosphate buffer
    150 mM sodium chloride
  2. Reduced osmium tetroxide
    1% osmium tetroxide (OsO4)
    1.5% potassium ferrocyanide
    0.1 M cacodylate buffer pH 7.4 (from sodium cacodylate)
    Note: Protect OsO4 from direct exposure to light during storage and use. OsO4 is highly toxic, handling and disposal in a fume hood are required.
  3. Sato lead solution
    0.21 g of lead citrate tribasic
    0.15 g lead nitrate
    0.15 g lead acetate trihydrate
    1 g of sodium citrate tribasic dehydrate
    Mix the chemicals and dissolve in 41 ml of distilled water. This makes a milky solution. Then, add 9 ml 4% NaOH and the solution will become clear. Filter the solution and store in a dark glass bottle at room temperature

Acknowledgments

Work in S.C. lab related to these topics is supported by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.) (IG 2015 Id.16721) and from European Commission-FP7-HPVAHEAD. DM was a Fondazione Italiana per la Ricerca sul Cancro (F.I.R.C.) fellow. The protocol is adapted from Mattoscio et al., 2017, and has been developed with the aid of the San Raffaele Institute Microscopy Facility ‘Alembic’. The authors declare no conflicts of interest.

References

  1. Eskelinen, E. L., Reggiori, F., Baba, M., Kovacs, A. L. and Seglen, P. O. (2011). Seeing is believing: the impact of electron microscopy on autophagy research. Autophagy 7(9): 935-956.
  2. Eskelinen, E. L., Prescott, A. R., Cooper, J., Brachmann, S. M., Wang, L., Tang, X., Backer, J. M. and Lucocq, J. M. (2002). Inhibition of autophagy in mitotic animal cells. Traffic 3(12): 878-893.
  3. Glick, D., Barth, S. and Macleod, K. F. (2010). Autophagy: cellular and molecular mechanisms. J Pathol 221(1): 3-12.
  4. Jäger, S., Bucci, C., Tanida, I., Ueno, T., Kominami, E., Saftig, P. and Eskelinen, E. L. (2014). Role for Rab7 in maturation of late autophagic vacuoles. J Cell Sci 117(Pt 20): 4837-48.
  5. Kabaeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y., and Yoshimori, T. (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19(21): 5720-8.
  6. Klionsky, D. J., Abdalla, F. C., Abeliovich, H., Abraham, R. T., Acevedo-Arozena, A., Adeli, K., Agholme, L., Agnello, M., Agostinis, P., Aguirre-Ghiso, J. A., Ahn, H. J., Ait-Mohamed, O., Ait-Si-Ali, S, Akematsu, T. and Akira, S., et al. (2016). Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12(1): 1-222.
  7. Mattoscio, D., Casadio, C., Miccolo, C., Maffini, F., Raimondi, A., Tacchetti, C., Gheit, T., Tagliabue, M., Galimberti, V. E., De Lorenzi, F., Pawlita, M., Chiesa, F., Ansarin, M., Tommasino, M. and Chiocca, S. (2017). Autophagy regulates UBC9 levels during viral-mediated tumorigenesis. PLoS Pathog 13(3): e1006262.
  8. Mizushima, N., Yoshimori, T. and Levine, B. (2010). Methods in mammalian autophagy research. Cell 140: 313-326.
  9. Schneider, J. L. and Cuervo, A. M. (2014). Autophagy and human disease: emerging themes. Curr Opin Genet Dev 26: 16-23.
  10. Stierhof YD, Humbel BM, Schwarz H. (1991). Suitability of different silver enhancement methods applied to 1 nm colloidal gold particles: an immunoelectron microscopic study. J Electron Microsc Tech 17(3): 336-43.
  11. Tokuyasu, K. T. (1980). Immunochemistry on ultrathin frozen sections. Histochem J 12(4): 381-403.
  12. Yla-Anttila, P., Vihinen, H., Jokitalo, E. and Eskelinen, E. L. (2009). Monitoring autophagy by electron microscopy in mammalian cells. Methods Enzymol 452: 143-164.

简介

尽管早在20世纪50年代就已经通过透射电子显微镜观察了自噬(在Eskelinen等人2011年的综述中),但是现在这种技术仍然是监测自噬过程的最强大的系统之一。 自噬体是包含细胞物质的LC3阳性双层膜结构,代表了自噬的关键细胞器,其简单的可视化和/或计数允许得出关于自噬流的重要结论。 因此,准确鉴定自噬体对自噬的全面和详细的分析至关重要。 在这里我们提出一个简单的协议,以确定autophagosomes透射电子显微镜耦合LC3的免疫金标记从一个相对较低的细胞数量,我们最近开发遵循病毒介导的人类癌变期间的自噬途径。

【背景】自噬体代表了macroautophagy的关键结构,这是一种细胞胞质成分的分解代谢系统。巨自噬(或简单地自吞噬)由吞噬细胞的形成引发,所述吞噬细胞能够自身扩张吞噬细胞器和蛋白质,所述蛋白质最终闭合在螯合成分周围形成被称为自噬体的细胞器。接下来,在成熟过程中,自噬体可以与溶酶体融合以形成自溶酶体,其中被捕获的物质被位于溶酶体限制性膜中的泵降解并再循环回(Glick et al。,2010)。

由于自噬功能障碍与各种人类疾病,病毒感染,神经退行性疾病,免疫功能和癌症有关(Schneider and Cuervo,2014),因此越来越需要对自噬作更全面和详细的分析。实际上,迄今为止已经开发了许多用于研究自噬的方法(Klionsky等人,2016),并且其中大多数方法需要同时进行以正确定义在特定实验中自噬是如何工作的系统。其中透射电子显微镜(TEM)结合特异性免疫金标记(免疫金电镜-IEM)仍然是自噬检测最准确的方法之一,因为它具有提供特定超微结构细节的精美能力。事实上,除了它们特有的形态(Yla-Anttila等,2009)之外,自噬体的特征还在于存在微管相关蛋白1轻链3或LC3,其通常被招募在自噬活化期间用磷脂酰乙醇胺(LC3-II)脂质化后的自噬膜上(Kabeya等人,2000)。因此,透射电镜结合抗LC3抗体标记细胞和与第二抗体偶联的金探针可以明确自噬体的识别,间接地可以监测自噬流。

培养细胞的免疫金标记通常使用几种方法:包埋前(树脂包埋前的免疫标记)和包埋后的树脂包埋后的免疫标记,包括Tokuyasu冷冻切片,其中冷冻保存的包埋标本首先通过浸入液氮然后在免疫标记之前用冷冻超薄切片机在-120℃切割(Tokuyasu,1980; Eskeline等人,2002; Jager等人,2004)。然而,两种技术都需要至少3×10 6个细胞来获得用于样品制备的足够的小球,从而推断在规划IEM实验期间初始细胞数量是一个关键因素。

因此,本文描述了从小细胞数(小于0.1×10 6个细胞)开始的用于自噬体检测的预包埋方案,其允许在具有低复制潜力的原代细胞中进行IEM分析。我们最近使用该方案在用与病毒癌变有关的逆转录病毒载体转导的原代人角质形成细胞群体上检测LC3阳性自噬体(Mattoscio等,2017)。原则上,该方法可用于检测其他感兴趣的抗原,因为我们也使用相同的方案来研究SUMO缀合酶UBC9的亚细胞定位(Mattoscio等人,2017),并且可以与不同的细胞种类一起使用,其初始细胞数目也是限制因素。

关键字:自噬, 免疫金电微镜技术, 自噬小体, LC3, 病毒致癌作用

材料和试剂

  1. 13mm Nunc Thermanox盖玻片(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:150067)
  2. 24孔组织培养处理板(Corning,Costar ,产品目录号:3524)
  3. Eppendorf Safe-Lock微量离心管(Sigma-Aldrich,目录号:T9661)
    制造商:Eppendorf,目录号:022363204。
  4. 边缘剃须刀刀片(西格玛奥德里奇,目录号:Z740504)
  5. 200目网格(Sigma-Aldrich,目录号:G5151)
  6. 张单层霉菌(电子显微镜科学,目录号:70920)
  7. 乙醇BioUltra,分子生物学(西格玛奥德里奇,目录号:51976)
  8. Dulbecco的磷酸盐缓冲盐水,钙,镁(Sigma-Aldrich,目录号:D8662)
  9. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
    注:为了制备4%多聚甲醛(PFA)溶液,在通风橱中在60℃下溶解4g PFA在90ml水中的溶液并滴加几滴1N NaOH(直到溶液不出现PFA沉淀物)然后取出加热,加入10毫升10×PBS,加水至100毫升,然后冷却。不要将溶液加热到70°C以上,因为PFA会在较高的温度下分解。立即使用溶液或在4°C或-20°C下保存1周。
  10. 戊二醛(Sigma-Aldrich,目录号:G7776)
  11. 淬灭溶液:在PBS中的50mM甘氨酸(Sigma-Aldrich,目录号:410225)
  12. 一抗:兔多克隆抗LC3(Novus Biologicals,目录号:NB100-2331)
  13. 洗涤缓冲液(0.1%BSA,0.1%皂苷,在PBS中)
  14. 与金结合的二级抗体:1.4nm纳米金IgG山羊抗兔IgG(Nanoprobes,目录号:2003)
  15. (增强剂),B(活化剂),C(引发剂)和D(缓冲剂)组成的金增强溶液(GoldEnhance TM TM EM)(Nanoprobes,目录号:2113) br />
  16. 醋酸铀酰(电子显微镜科学,目录号:22400)
    注意:醋酸铀酰具有轻微的反应性并且通过摄取具有高毒性。请根据您的机构政策处理废物。
  17. Embed-812树脂套件(电子显微镜科学,目录号:14120)
    注意:
    1. 这款产品已经停产,取而代之的是EMbed 812。
    2. 为了更好地保存,将Embed-812(32g)与十二碳烯基琥珀酸酐(DDSA)(16g)和NMA(Nadic Methyl Anhydride)(17.4g)混合。充分混合,加入0.8克促进剂DMP30(2,4,6-三(二甲氨基甲基)苯酚),混合并储存于-20℃的20毫升注射器中。在使用前加热至室温。
  18. 氢氟酸(Fisher Scientific,目录号:14-650-236)
    制造商:Avantor Performance Materials,J.T.贝克,目录号:9560-01。
  19. 阻塞缓冲区(见食谱)
    1. 牛血清白蛋白(BSA,无IgG和无蛋白酶)(Jackson ImmunoResearch,目录号:001-000-162)
    2. 正常山羊血清(Jackson ImmunoResearch,目录号:005-000-121)
    3. 氯化铵(Sigma-Aldrich,目录号:A9434)
    4. 皂苷(Sigma-Aldrich,目录号:47036)
    5. 磷酸盐缓冲液(Sigma-Aldrich,目录号:P3619)
    6. 氯化钠(Sigma-Aldrich,目录号:S7653)
  20. 减少四氧化锇(见食谱)
    1. 四氧化锇(OsO 4)(Electron Microscopy Science,目录号:19140)
    2. 亚铁氰化钾(电子显微镜科学,目录号:20150)
    3. 二甲胂酸盐缓冲液pH 7.4(来自二甲胂酸钠三水合物)(Sigma-Aldrich,目录号:C4945)
  21. 佐藤铅解决方案(见食谱)
    1. 铅柠檬酸三钠(Sigma-Aldrich,目录号:15326)
    2. 硝酸铅(默克,目录号:1073980100)
    3. 柠檬酸三钠脱水物(Sigma-Aldrich,目录号:S4641)

设备

  1. 通风柜
  2. 60°C烤箱(Memmert,型号:UN30)
  3. 超微切片机(Leica Microsystem,型号:Leica EM UC7)
  4. 透射电子显微镜(卡尔•蔡司,型号:Leo 912AB)配备慢扫Proscan相机(ProScan)
  5. 摇摆器

程序

  1. 固定术
    1. 用乙醇消毒盖玻片,用PBS洗三次,并为24孔板的每个孔容纳一个载玻片。
    2. 用适当的培养基将这些载玻片铺在这些载玻片上,第二天获得60-70%的融合度。
      注:为了获得足够的技术重复,我们通常为每个条件准备6张幻灯片进行测试。在我们的研究中,我们使用表达空对照载体的原代角质形成细胞或用于HPV癌基因蛋白E6 / E7的过表达的质粒(以约1×10 5个细胞/孔接种)(Mattoscio et al。 ,2017)。此外,为了明确识别自噬体,应制备自噬体形成的适当的阳性和阴性对照。例如,考虑用雷帕霉素10-500nM(以增加自噬体形成)或用III类PI3K抑制剂(诸如1-10mM 3-甲基腺嘌呤或10-100nM渥曼青霉素以抑制自噬体积累)激活/抑制自噬(分别为Mizushima等人,2010)。
    3. 第二天,取出培养基,用PBS洗涤2次,并在室温下将细胞在4%新鲜制备的PFA在0.1M磷酸盐缓冲液pH7.4中固定1小时。
      注意:由于戊二醛降低了蛋白质的抗原性,虽然它提供了最好的结构保存,但我们宁愿先用PFA单独固定细胞,然后在抗原标记后用戊二醛重新固定。
    4. 去除PFA,用PBS清洗2次。将固定的细胞储存在4°C。
      注意:由于PFA固定是可逆的,因此长时间储存将细胞保存在储存溶液(PBS中的1%PFA)而不是单独的PBS中。

  2. 免疫标记
    1. 取出固定液或储存液,每孔用PBS冲洗3次,每次5分钟,室温下用淬火液处理10分钟,淬灭主要固定剂的游离醛。
    2. 通过在室温下用0.25%皂角苷,0.1%BSA的PBS溶液孵育10分钟使细胞透化。
    3. 用封闭缓冲液(0.2%BSA,5%正常山羊血清,50mM NH 4 Cl,0.1%皂角苷,20mM磷酸盐缓冲液,150mM NaCl,室温下孵育细胞30分钟,参见食谱)阻止非特异性染色。
    4. 去除封闭缓冲液,在封闭缓冲液中孵育盖有抗LC3一抗(1:150稀释)的盖玻片,在室温下孵育2小时。
    5. 用洗涤缓冲液(0.1%BSA,0.1%皂苷的PBS溶液)洗涤细胞4次,每次5分钟。
    6. 在封闭缓冲液中孵育含有与纳米金(1:200稀释)缀合的1.4nm纳米金羊山羊抗兔二抗的盖玻片1小时。
    7. 用洗涤缓冲液洗3次,用PBS洗3次。
    8. 用含1%戊二醛的PBS在室温下重新固定染色的细胞30分钟以保持超微结构。冲洗在PBS中,并在4°C的PBS中,直到下一步。

  3. 黄金增强

    1. 在室温下平衡黄金增强溶液(A,B,C,D)的所有成分
    2. 用PBS中的50mM甘氨酸洗3次5分钟。
    3. 用1%BSA,0.05%Tween的PBS溶液洗3次5分钟。
    4. 在PBS中冲洗3次,同时通过在Eppendorf管中加入一份各自的组分(A增强剂和B激活剂)激活金增强溶液,充分混合并孵育5分钟。
      注意:黄金增强溶液应在使用前立即准备。增加组分B的量可能有助于减少非特异性信号。
    5. 加入黄金增强混合物的一部分组分C(引发剂)和D(缓冲液),分别在蒸馏水中冲洗细胞,并添加数滴的解决方案盖玻片。每盖玻片准备约40微升黄金增强解决方案。
    6. 检查深紫色/黑色沉淀物的外观(图1)。黄金增强解决方案的孵化时间可能会有所不同。通常情况下,需要1到3分钟的时间才能完成开发。总是比较阴性对照(没有一抗)。
      注意:这一步骤的目标是扩大与二抗相关的金颗粒。为了改善渗透和密度标记,小尺寸的金颗粒是优选的(1.4nm)。由于这些纳米金颗粒在样品中几乎不可见,所以这一步是强制性的。但是,作为黄金的替代品,可以使用银色增强解决方案。银增强剂都是可商购的(Nanoprobes,目录号2013)或可以由单一组分制备(Stierhof等,1991)。银增强是光敏感的,可以被四氧化锇破坏,因此黄金增强溶液通常是首选的,因为它的简单使用。

    7. 每次用蒸馏水洗三次,每次5分钟

      图1.黄金增强。在盖玻片上放一滴黄金增强溶液,并孵化,直到出现黑暗的沉淀物(肉眼可见)。较长的孵化可能会导致较暗的信号。在明亮的现场显微镜或立体显微镜下进行检查,以便对黄金开发进行精细控制。请注意,暗沉淀不会出现在阴性对照(CTRL:无一抗)。底部:在时间= 0和金增强孵育结束时(T = 3分钟)放大盖玻片。请注意,黑色沉淀只出现在第一抗体的地方。

  4. 对比度增强
    1. 后处理样品与四氧化锇减少(见食谱)1小时在RT。
      在0.1M二甲胂酸钠中洗3次,每次5分钟,用蒸馏水洗三次 注:使用减少锇固定步骤保留膜脂质。亚铁氰化钾的加入减少了锇,使其对膜更具反应性,提高了它们的对比度。
    2. 在4℃下用1%乙酸双氧铀水溶液整体染色过夜。
      注意:不要暴露在光线下。由于铀酰和铅离子对碳水化合物和RNA中的蛋白质,核酸和羟基的高亲和力,醋酸铀酰提高了图像对比度。使用水代替酒精溶液更好地保存细胞结构是很重要的。

  5. 嵌入
    1. 在升序排列的乙醇系列中,每个升序排列30%,50%,70%,80%,90%,96%的样本5分钟。最后,在100%乙醇中每次脱水3次10分钟。

    2. 用乙醇包埋液1:1的无水乙醇溶液置于摇床上2小时。
    3. 从乙醇树脂混合物中取出盖玻片,用纸巾吸取多余的树脂,并将盖玻片放在一滴树脂上面。 1小时后,将盖玻片移到新的树脂滴(图2A)。


      图2.嵌入。在树脂滴上移动盖玻片,孵育1小时(A)。将一滴Epon放在张氏模具上,盖上盖玻片(B)。

    4. 用几滴树脂填充张单层模具的腔体,并在腔体的顶部放置盖玻片(确保清除盖玻片上面的多余树脂)(图2B)。

    5. 在45°C烘箱中过夜,然后升温至60°C,再烘24小时。

  6. 切割
    1. 要从幻灯片上分离Epon,请将Epon块从烤箱中取出,让其冷却下来。然后,在液氮中浸泡30秒,然后浸入热水中。这将从最初生长细胞的盖玻片上分离含有嵌入细胞的树脂。
      注意:或者,将盖玻片放入塑料多孔,盖上浓氢氟酸溶液(30%),摇动5-10分钟,直到玻璃完全溶解。
    2. 用蒸馏水洗涤样品数次,将样品浸泡在0.1M二甲胂酸盐缓冲液中30分钟,然后用蒸馏水洗涤并干燥。
    3. 使用单刃剃须刀去除树脂块的一小部分,并在空的树脂块上粘上氰基丙烯酸酯胶(图3)。


      图3.截面。在玻璃盖玻片被移除后,用一把剃刀刀片剪下一小块树脂块,并粘在一个空的树脂块(橡皮泥,图中的蓝色材料)的顶部。 1-2小时后,可将样品放入超薄切片机中切片。

    4. 切割厚度为60-90纳米的超薄切片,并将切片收集到具有正方形或六边形开口的200目网格上。
    5. 用1%醋酸铀水溶液对网格进行5分钟的染色,然后用佐藤的铅溶液(参见食谱),染色2分钟。
    6. 在电子显微镜下观察并拍摄数码照片。
      注意:为了使自噬体可视化,应当以放大4,000倍的放大率(总览)至10,000倍(精细观察超微结构)观察样品,寻找LC3金颗粒阳性的双膜细胞器,并且含有未消化的细胞质内容物图4A)。

数据分析

在这个协议中,我们采用了预嵌入技术(免疫标记之前,样品嵌入树脂),以检测LC3在低丰度细胞的制备。我们最近使用这种方法在表达空载体或HPV E6 / E7癌基因蛋白的原代人角质形成细胞中对UBC9和LC3阳性自噬体进行成像(Mattoscio et al。,2017)。事实上,TEM分析显示UBC9存在于双膜细胞内结构中,我们通过LC3免疫金TEM鉴定为自噬区室,提示UBC9降解的自噬依赖性途径。 
尽管TEM是评估自噬过程的最佳技术之一,但自噬是一个动态过程的事实使得难以从单个超微结构快照推断有意义的数据。而且,由于与溶酶体融合发生的降解过程,一些晚期的自噬结构难以鉴定。在图4中,我们报道了人类角质形成细胞中自噬结构的形态(用箭头表示),与经典的EM制备(左)和LC3免疫标记(右)并排比较,说明了我们的策略获得的典型结果。尽管保留了关于细胞结构的详细信息(图4),并且使用抗LC3抗体标记细胞使得能够明确识别自噬,但是初始的轻度固定与单独PFA的组合和预包埋染色允许更好的抗原保存空泡。此外,直接在显微镜载玻片上固定细胞而不需要刮擦和沉淀细胞允许以相对低的细胞数工作并维持细胞超微结构。因此,我们相信这个协议可以很容易地应用于检测罕见细胞群中额外的低丰度抗原。然而,这种方法的局限性在于它不能进行共定位研究,这只能通过使用不同大小的金探针进行嵌入后EM来实现。


图4.使用TEM和LC3免疫染色的自噬体识别A.左:原代人角质形成细胞的TEM图像。右:透射电镜结合LC3免疫金标记。箭头表示自噬空泡。请注意,金颗粒(LC3)选择性富集自噬结构。 B.图4A中圈出的自噬泡的放大倍数(右)。注意膜结构,LC3阳性,并与溶酶体(较暗的身体)融合。 

食谱

  1. 阻塞缓冲区
    0.2%牛血清白蛋白(BSA,无IgG和无蛋白酶)
    5%正常山羊血清
    50 mM氯化铵
    0.1%皂苷
    20 mM磷酸盐缓冲液
    150 mM氯化钠
  2. 减少四氧化锇
    1%四氧化锇(OsO 4)
    1.5%亚铁氰化钾
    0.1M二甲胂酸盐缓冲液pH7.4(来自二甲胂酸钠)
    注意:在储存和使用过程中,避免OsO4直接暴露于光线下。 OsO4毒性很强,需要在通风橱中处理和处置。
  3. 佐藤领导的解决方案
    0.21克柠檬酸铅三价铬
    0.15克硝酸铅
    0.15克醋酸铅三水合物
    1克柠檬酸三钠脱水
    混合化学物质并溶于41毫升蒸馏水中。这使得乳白色的解决方案。然后,加入9毫升4%的氢氧化钠溶液将变得清澈。过滤溶液并在室温下储存在黑色玻璃瓶中

致谢

在S.C.实验室中与这些主题相关的工作得到意大利商业协会(A.I.R.C.)(IG 2015 Id.16721)和欧盟委员会FP7-HPVAHEAD的支持。 DM是一名意大利人,每个人都是RiccíaS.Cancro(F.I.R.C.)的学者。该协议改编自Mattoscio等人,2017年,并在San Raffaele研究所显微设备“Alembic”的帮助下开发。作者宣称没有利益冲突。

参考

  1. Eskelinen,E.L.,Reggiori,F.,Baba,M.,Kovacs,A.L。和Seglen,P.O.(2011)。电子显微镜对自噬研究的影响看到相信自噬研究。 自噬 7(9):935-956。
  2. Eskelinen,E.L.,Prescott,A.R.,Cooper,J.,Brachmann,S.M.,Wang,L.,Tang,X.,Backer,J.M。和Lucocq,J.M。(2002)。 抑制有丝分裂动物细胞中的自噬 Traffic 3 (12):878-893。
  3. Glick,D.,Barth,S.和Macleod,K.F。(2010)。 自噬:细胞和分子机制
  4. Jäger,S.,Bucci,C.,Tanida,I.,Ueno,T.,Kominami,E.,Saftig,P.和Eskelinen,E.L。(2014)。 Rab7在晚期自噬泡的成熟中的作用 J Cell Sci 117(Pt 20):4837-48。
  5. Kabaeya,Y.,Mizushima,N.,Ueno,T.,Yamamoto,A.,Kirisako,T.,Noda,T.,Kominami,E.,Ohsumi,Y.和Yoshimori,T。(2000)。 LC3是酵母Apg8p的哺乳动物同源物,在加工后定位于自噬体膜中。 / em> EMBO J 19(21):5720-8。
  6. Klionsky,DJ,Abdalla,FC,Abeliovich,H.,Abraham,RT,Acevedo-Arozena,A.,Adeli,K.,Agholme,L.,Agnello,M.,Agostinis,P.,Aguirre-Ghiso,JA, Ahn,HJ,Ait-Mohamed,O.,Ait-Si-Ali,S,Akematsu,T。和Akira,S。等人。 (2016)。 使用和解释自噬监测分析指南(第三版) 自噬 12(1):1-222。
  7. Mattoscio,D.,Casadio,C.,Miccolo,C.,Maffini,F.,Raimondi,A.,Tacchetti,C.,Gheit,T.,Tagliabue,M.,Galimberti,VE,De Lorenzi,F., Pawlita,M.,Chiesa,F.,Ansarin,M.,Tommasino,M.和Chiocca,S。(2017)。 自噬调节病毒介导的肿瘤发生过程中的UBC9水平 PLoS Pathog 13(3):e1006262。
  8. Mizushima,N.,Yoshimori,T.和Levine,B。(2010)。 哺乳动物自噬研究方法 140:313 -326。
  9. Schneider,J.L。和Cuervo,A.M。(2014)。 自噬与人类疾病:新兴主题 em> 26:16-23。
  10. Stierhof YD,Humbel BM,Schwarz H.(1991)。 适用于1纳米胶体金颗粒的不同银增强方法的适用性:免疫电子显微镜研究。 J Electron Microsc Tech 17(3):336-43。
  11. Tokuyasu,K.T。(1980)。 超薄冰冻切片的免疫化学
  12. Yla-Anttila,P.,Vihinen,H.,Jokitalo,E.和Eskelinen,E.L。(2009)。 通过电子显微镜在哺乳动物细胞中监测自噬方法Enzymol 452:143-164。
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引用:Mattoscio, D., Raimondi, A., Tacchetti, C. and Chiocca, S. (2017). Immunogold Electron Microscopy of the Autophagosome Marker LC3. Bio-protocol 7(24): e2648. DOI: 10.21769/BioProtoc.2648.
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