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Proximal Ligation Assay (PLA) on Lung Tissue and Cultured Macrophages to Demonstrate Protein-protein Interaction
对肺组织和培养的巨噬细胞进行邻位连接分析(PLA)以验证蛋白质 - 蛋白质相互作用   

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Abstract

In this protocol, we describe proximal ligation assay (PLA), an antibody-based detection method for protein-protein interaction. This method relies on specific binding of individual primary antibodies to the two putative interacting proteins. The primary antibodies need to have different hosts. The secondary antibodies against the two hosts have complementary oligonucleotide moieties attached to them. If the two antigens are in close proximity (presumably interacting with each other), the complementary oligonucleotides can anneal and fluorescent nucleotides can be incorporated in a single DNA polymerization step. Under a microscope, these reactions appear as punctate fluorescent spots, indicating successful PLA reaction and suggesting protein-protein interaction between the two antigens.

Keywords: Proximal ligation assay(邻位连接分析), Protein-protein interaction(蛋白质-蛋白质相互作用), Toll-like receptors(Toll样受体), Fluorescence microscopy(荧光显微镜检查), Bronchial epithelium(支气管上皮), Macrophages(巨噬细胞), J774(J774), Duolink(Duolink)

Background

Proximal ligation assay (PLA) is an antibody-based technique to determine whether two proteins are with 40 nm of each other. Proteins detected in this manner are identifiable by fluorescence (Ho et al., 2012; Banerjee et al., 2015). This makes PLA an excellent tool for locating protein-protein interactions. Activation of toll-like receptor (TLR) pathways is an important part of the innate immune response to pathogenic threats. TLRs recognize pathogen-associated molecules and induce a signaling cascade to effect a rapid response to infection. TLR2 and TLR4 are two well-studied members of the TLR family that respond to different stimuli. While both receptors activate in response to bacterial infection, only TLR4 responds to lipopolysaccharide exposure. They activate some shared signaling cascades, however, including the MyD88/Traf6 pathway. The induction of this pathway includes the formation a signaling complex known as the myddosome (Gay et al., 2011; Xiong et al., 2011; Cleaver et al., 2014), a protein complex that includes the MyD88, IRAK1, IRAK4 and Traf6 among others. Myddosome assembly results in NF-κb-mediated inflammatory response and pathogen clearance.

Visualizing the engagement of TLR signaling pathways is an important step in identifying and locating immune response. Here, we use PLA to detect TLR pathway activation in fixed lung tissue and a cultured peritoneal macrophage cell-line under treatment with LPS or exposure to opportunistic infection. This method fluorescently labels proteins that interact and remain within close proximity. Using fluorescence microscopy to visualize the resulting labels in vivo allows us to identify the protein complex in respect to tissue location. Here, we demonstrate the ability of this assay to detect TLR2 activation during opportunistic lung infection in vivo and myddosome formation after LPS treatment of peritoneal macrophage cells in vitro. We have also shown the specificity of the technique, as it does not indicate TLR2 activation after LPS treatment in vivo.

Materials and Reagents

  1. NuncTM Lab-TekTM II Chamber SlideTM System (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 154534 )
  2. 0.22 µm PVDF syringe filter (EMD Millipore, catalog number: SLGV004SL )
  3. Coverslip (22 x 40 mm) (VWR, catalog number: 470019-008 )
  4. Lung tissue harvested from morphine-treated and/or Streptococcus pneumoniae-infected mice
  5. J774 cells, a murine peritoneal macrophage cell line (ATCC, catalog number: TIB-67 )
  6. Primary antibodies
    Note: Each pair used in PLA reaction needs to be validated for specificity with Western blot (both native and SDS-PAGE) and must come from a different host, compatible with Duolink secondary antibodies with PLA probes.
    1. Affinity isolated anti-TRAF6 antibody raised in rabbit (Sigma-Aldrich, catalog number: SAB2102531 )
      Note: This product has been discontinued.
    2. Affinity isolated anti-TLR2 antibody raised in rabbit (Sigma-Aldrich, catalog number: SAB1300199 )
    3. Monoclonal anti-MYD88 (Clone OTI2B2) (OriGene Technologies, catalog number: TA502117 )
  7. Secondary antibodies linked to PLA probes
    1. Duolink® in situ PLA® probe anti-mouse MINUS, Affinity purified Donkey anti-Mouse IgG (H+L) (Sigma-Aldrich, catalog number: DUO92004 )
    2. Duolink® in situ PLA® probe anti-rabbit PLUS, Affinity purified Donkey anti-Rabbit IgG (H+L) (Sigma-Aldrich, catalog number: DUO92002 )
  8. Formalin solution, neutral buffered, 10% (10% NBF) (Sigma-Aldrich, catalog number: HT501128-4L )
  9. Paraffin (Fisher Scientific, catalog number: P31-500 )
  10. Xylene (Histological grade) (Fisher Scientific, catalog number: X3S-4 )
  11. Ethanol 200 proof (Merck, catalog number: AX0441 )
  12. Fixation-permeabilization buffer set (Thermo Fisher Scientific, eBiosciencesTM, catalog number: 88-8824-00 )
  13. Phosphate-buffered saline (PBS) pH 7.4 (1x) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  14. Tween® 20 (Fisher Scientific, catalog number: BP337-100 )
  15. Duolink® in situ mounting medium with DAPI (Sigma-Aldrich, catalog number: DUO82049 )
  16. Nail polish (as cover slip sealant)
  17. DMEM/High glucose (4,500 mg/L L-glucose) (GE Healthcare, HyCloneTM, catalog number: SH30243.01 )
  18. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  19. Fetal bovine serum (FBS), qualified, USDA-approved regions (Thermo Fisher Scientific, GibcoTM, catalog number: 10437010 )
  20. Ultrapure 0.5 M EDTA, pH 6.0 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575020 )
  21. Lipopolysaccharides from Escherichia coli O127:B8 (Sigma-Aldrich, catalog number: L3880 )
  22. Duolink® in situ wash buffers, fluorescence (Sigma-Aldrich, catalog number: DUO82049 )
  23. Duolink® in situ detection reagents orange (Sigma-Aldrich, catalog number: DUO92007 )
  24. Duolink in situ wash buffers A and B (Sigma-Aldrich, catalog number: DUO82047 )
  25. Duolink in situ wash buffer A working solution (see Recipes)
  26. Duolink in situ wash buffer B working solution (see Recipes)

Equipment

  1. Coplin Jar (Generic)
  2. Oil marker (Aqua-hold pap pen) (Electron Microscopy Sciences, catalog number: 71311 )
  3. Vegetable steamer (Generic)
  4. Slide humidity chamber (Simport, model: M920 )
  5. Laboratory shaker or rocker
  6. Flourescence filters (Leica)
    1. Filter set 49, Excitation G365, Emission 445/50
    2. Filter set 43 HE, Excitation BP550/25, Emission 605/70
  7. Flourescence microscope

Software

  1. ImageJ software (https://imagej.nih.gov/ij/)

Procedure

  1. Antibody selection
    1. To identify protein-protein interactions, primary antibodies recognizing both proteins must be used. Since the specificity of the antibody is of paramount importance, it is recommended that the antibody is used for Western blot analysis using tissue lysate in both naïve and denaturing conditions. The goal is to obtain highest specificity in both conditions as the following steps might have a mix of target antigens in naïve and denatured condition within the cell.
    2. Primary antibodies must differ in the source species (e.g., anti-MyD88 raised in rabbit and anti-TRAF6 raised in mouse).
    3. Each oligonucleotide-conjugated secondary antibody must recognize one of the primary antibodies.

  2. Sample preparation
    1. Tissue slides
      1. Tissue collected from in vivo sources should be immediately fixed in 10% NBF before embedding in paraffin.
      2. Mount 5-8 µm sections from paraffin blocks to slides.
      3. Deparaffinize slides by dipping them into xylene bath (3-4 dips and 15-20 sec holding time) and rehydrate slides with serial ethanol washes (100, 70, 50, 30 and 0% solutions with water).
      4. Add permeabilization buffer (1x) sufficient to cover the section for 40 min at 4 °C in the dark.
      5. Wash the slides once with PBS and incubate slides in a vegetable steamer for 1 h.
      Note: In our example, we use lung tissue harvested from morphine-treated and/or pneumococcus-infected mice.
    2. Cultured cell slides
      1. Plate 40,000 to 50,000 cells/ml media, on a chamber slide and incubate overnight to allow cells to adhere to the slide to a concentration of 60-80% confluence.
      2. Perform any necessary treatments to the cells within chambers.
      3. Remove media and immediately fix cells by adding 10% NBF to each chamber and incubating for 20 min at room temperature.
      4. Remove chamber walls.
      5. Wash slides with PBS three times.
      6. Permeabilize by incubating slides with 0.01% Tween 20 in PBS for 10 min at room temperature.
      7. Wash slides with PBS three times.
      Note: In our example, we use J774 cells, a murine peritoneal macrophage cell line. We treated these cells with different concentrations of LPS for 1 h to induce myddosome ligation with TLR4.

  3. PLA protocol
    1. Tap slides to remove excess liquid then air-dry and outline tissue sections or wells with an oil marker.
    2. Incubate slides with blocking solution in a humidity chamber for 30 min at 37 °C.
    3. Remove blocking solution and add enough of both primary antibodies reconstituted in blocking solution (approximately 1 µg antibody/chamber or per section) to cover each section or chamber.
      Note: Approximately 40 µl of solution will be sufficient to cover a chamber.
    4. Incubate in a humidity chamber overnight at 4 °C.
    5. Place slides in a Coplin jar filled with wash buffer A (see Recipes) two times for five minutes each on a laboratory shaker.
    6. Add oligonucleotide-linked secondary antibody solution, diluted 1:10 in wash buffer A (enough to cover the section/cells) to slides.
    7. Incubate in a humidity chamber for 1 h at 37 °C.
    8. Wash in wash buffer A two times for five minutes each on a shaker.
    9. Add ligase solution to slide (enough to cover the section/cells) and incubate for 30 min in a humidity chamber at 37 °C.
      Note: Working concentration of ligase should be 1:40 from the original vial, diluted with molecular biology grade water.
    10. Wash in wash buffer A two times for five minutes each on a shaker.
    11. Add premixed polymerase and nucleotide solution to slide and incubate for 100 min at 37 °C.
      Note: After this point, limit exposure of slide to light. Working concentration of polymerase should be 1:80 of original solution, diluted with molecular biology grade water. Wash in wash buffer B (see Recipes) twice for 10 min each.
    12. Wash in 1x wash buffer B for one minute.
    13. Allow slides to dry.
    14. Mount coverslip in mounting medium with DAPI.
    15. Seal edges of cover slip with nail polish.
    16. Allow to dry.

Data analysis

  1. Imaging (Figure 1)
    1. Using a fluorescence microscope and DAPI-appropriate filter (such as Filter set 49 from Leica), identify and photograph cells/tissue on your slide.
    2. Photograph the same area using a filter appropriate for your PLA probe.
      Notes:
      1. In our example, we use filter set 43 HE from Leica to identify our PLA probe, Duolink in situ detection reagents orange.
      2. Be sure to save the individual image files for your DAPI and PLA probe images separately, in addition to the merged image.


        Figure 1. Use of ImageJ to detect fluorescent particles. Color threshold was used as described above. A. Unedited image of DAPI-stained J774 cells; B. The same image after application of a color threshold; C. Example of the settings used for this threshold.

  2. Quantification (Figure 2)
    1. Determine cell count
      1. In ImageJ, open the file containing the DAPI image.
      2. Select Image > Adjust > Color Threshold...
        Note: This applies to color images. If using a grey-scale image, select Image > Adjust > Threshold...
      3. Adjust the parameters in this dialog box until DAPI-stained nuclei are entirely, but distinctly highlighted.
      4. Select Analyze > Analyze particles...
      5. Check the ‘Summary’ checkbox then select ‘OK’.
      6. The resulting ‘Summary’ window will contain the total count of nuclei.
    2. Determine the number of PLA reactions
      1. In Image J, open the file containing the PLA image.
      2. Select Image > Adjust > Color Threshold...
        Note: This applies to color images. If using a grey-scale image, select Image > Adjust > Threshold...
      3. Adjust the parameters in this dialog box until PLA reaction sites are entirely, but distinctly highlighted.
      4. Select Analyze > Analyze particles...
      5. Check the ‘Summary’ checkbox then select ‘OK’.
      6. The resulting ‘Summary’ window will contain the total count of PLA reactions.
    3. Compare the number of PLA reactions in your sample to the number of nuclei to determine the number of protein interactions per cell.
      Notes: This is a simple ratio of positive PLA fluorescence and DAPI.
      1. Macros and batch analysis may be used to rapidly analyze multiple pictures in ImageJ.
      2. If the cell number is low in the image, manual counting may be better suited and more accurate for your study.


        Figure 2. Example of a, IJM language macro used to create a color threshold for PLA reactions in a fluorescent image. Because the image used was only the PLA fluorescence instead of the merged image, min and max settings for the hue and saturation could be inclusive (Lines 14, 15 for Hue; lines 17, 18 for saturation). Minimum brightness was adjusted to eliminate background while including signal (Line 20). Macro creates a mask and counts particles remaining after the threshold is applied. Macro may be used to count particles from multiple image files at once by use of the batch function built into ImageJ.

  3. Results
    1. J774 cells: J774 is a murine macrophage cell lines, which was treated with varying concentrations of LPS to activate the TLR4 pathway. This entails recruitment of Myd88 (as a part of a bigger myddosome complex) to TLR4 and its subsequent ligation to Traf6, an E3 ubiquitin ligase. If TLR4 is activated successfully, Myd88 and Traf6 would be ligated within the first hour of reaction as shown in Figure 3.


      Figure 3. Proximal ligation assay showing MyD88 Traf6 activation in J774 cells. Cells were treated with LPS at varying concentrations for 1 h before fixation and PLA. A. Untreated J774 cells; B. 2 ng/ml LPS-treated J774 cells; C. Quantification of the number of ligation reactions per DAPI-stained cell. Scale bars =100 µm.

    2. Lung Section: C57bl6/j animals were either treated with Morphine or S. pneumoniae or both for 72 h and their lung sections were used to perform PLA between TLR2 and Myd88. As shown in Figure 4, individual treatments did not exhibit positive PLA reaction. This is expected because morphine treatment by itself does not activate TLRs, but makes the individual susceptible to opportunistic infections. S. pneumoniae alone also doesn’t activate TLR2 due to intact epithelial barrier of the lungs. A combined treatment, however, shows positive PLA reaction due to activation of TLR2.


      Figure 4. PLA showing TLR2/MyD88 interaction in mouse lung epithelium. Images represent (A) 72-h morphine treatment (B) Infection with S. pneumoniae (C) 72-h morphine treatment and S. pneumoniae infection. D. Expanded section showing ligation reaction sites (Arrows) in the lung epithelium of the Morphine + Pneumococcus group. E. Results were quantified by counting the number of ligation reactions per DAPI-stained nucleus. Scale bars = 100 µm.

    3. Negative control: This experiment was performed to test the integrity of the method, in addition to individual controls within each experiment. As shown in Figure 5, we performed a PLA reaction for TLR2 and Myd88 in J774 cells treated with LPS. This reaction was negative as expected, since LPS stimulates TLR4 and Myd88 should not bind to TLR2 under these conditions.


      Figure 5. PLA showing no interaction between TLR2 and MyD88 in J774 cells treated with LPS. We observed few or no PLA reactions in any of the treatment groups. LPS is an activator of TLR4, not TLR2. While both TLR2 and MyD88 antibodies are likely interacting with their respective antigens, the antigens are not present within 40 nm of each other and will not induce PLA reactions. Scale bars = 100 µm.

    4. Positive control: We validated the capability of the method to detect protein interactions. As shown in Figure 6, we performed a PLA reaction for TLR4 and CD14 in J774 cells treated with LPS. We detected LPS-dependent interaction between TLR4 and CD14. This is expected since TLR4/CD14 interaction under LPS treatment has been characterized thoroughly.


      Figure 6. Proximal ligation assay showing TLR4/CD14 interaction in J774 cells treated for 30 min with LPS. A. Untreated J774 cells; B. 0.4 ng/ml LPS-treated J774 cells; C. 2 ng/ml LPS-treated J774 cells; D. Quantification of the number of ligation reactions per DAPI-stained cell. Scale bars = 50 µm.

Notes

As mentioned before, Duolink PLA method works reproducibly well in both in vitro and in vivo conditions with caveats. The considerations one should keep in mind are as follows:

  1. In our experience, the quality of chamber slides is very important. Various brands were tried and Nunc brand (No. 1 in Materials above) gave us the cleanest results. Chamber slides with synthetic base should be avoided. Good quality optically clear glass base works the best.
  2. We have used the 8-well chamber slide for reagent optimization and having enough experimental replicates (n = 4 in this case for each condition). However, care should be taken in washing steps since the edge surface tension is quite strong in these chamber slides and optimal wash could be challenging. While washing, occasional swirling motion is advised to cover the whole chamber, including the edges.
  3. PLA works best for 60-80% confluent cells. We have always preferred this concentration since it leaves patches of the chamber base empty, which serves as an additional control for non-specific binding of the reagents.
  4. For in vivo experiments, the background staining is higher than in plated cells. This is expected and the non-specific background is usually a uniform hue, rather than punctate stain. Due to high levels of non-specific staining observed in in vivo samples, you may wish to increase blocking times for tissue samples. This should be optimized depending on the source of the tissue e.g., intestinal and adipose tissues may require less or more incubation time compared to the lung tissues used here.
  5. Fixed tissue slides must be subject to antigen retrieval before incubation with individual antibodies. A combination of flow-cytometric permeabilization buffer and vegetable steamer based antigen retrieval has consistently given us successful PLA reaction.
  6. In general, PLA reaction always gives punctate fluorescence. In rare circumstances, if the PLA reaction gives continuous stain, specific reaction should be verified using proper experimental controls and single-antibody controls.
  7. In reference to the selection of primary antibodies, each pair used in the PLA reaction needs to be validated for specificity by Western blot (both native and SDS-PAGE) and must come from a different host, compatible with Duolink secondary antibodies with PLA probes.

Recipes

  1. Duolink in situ wash buffer A
    1. Alternative 1–use Duolink in situ wash buffer A
      To prepare a 1x buffer, dissolve the content of one pouch in high purity water to a final volume of 1,000 ml. Store pouches at room temperature
      Note: Expiry date is marked on each individual lot. 1x solutions may be kept at room temperature for short time storage (one week or less). For long time storage store at 4 °C. Bring the solutions to room temperature before use.
    2. Alternative 2–make your own Duolink in situ wash buffer A
      1. Dissolve 8.8 g NaCl, 1.2 g Tris base and 0.5 ml Tween 20 in 800 ml high purity water
      2. Adjust pH to 7.4 using HCl
      3. Add high purity water to 1,000 ml (final concentrations 0.01 M Tris, 0.15 M NaCl and 0.05% Tween 20)
      4. Filter the solution through a 0.22 µm filter and store at 4 °C
      5. Bring the solutions to room temperature before use
  2. Duolink in situ wash buffer B
    1. Alternative 1–use Duolink in situ wash buffer B
      To prepare a 1x buffer, dissolve the content of one pouch in high purity water to a final volume of 1,000 ml. Store pouches at room temperature
      Note: Expiry date is marked on each individual lot. 1x solutions may be kept at room temperature for short time storage (one week or less). For long time storage store at 4 °C. Bring the solutions to room temperature before use.
    2. Alternative 2–make your own Duolink in situ wash buffer B
      1. Dissolve 5.84 g NaCl, 4.24 g Tris base and 26.0 g Tris-HCl in 500 ml high purity water
      2. Adjust pH to 7.5 using HCl
      3. Add high purity water to 1,000 ml (final concentrations 0.2 M Tris and 0.1 M NaCl)
      4. Filter the solution through a 0.22 µm filter and store at 4 °C
      5. Bring the solutions to room temperature before use

Acknowledgments

This work was partially supported by NIH R21HL125021 and University of Miami institutional support (to SB). The authors declare no conflict of interests. Data for all figures were generated exclusively for this protocol. However, PLA protocol was adapted for J774 cells from our previous publication (Banerjee et al., 2015), SciRep 5:11384; Reference No. 1 for this protocol), where it was performed on 16HBE14o (human bronchial epithelium cell line). PLA on lung tissue was adapted and modified exclusively for this protocol as described in the text.

References

  1. Banerjee, S., Ninkovic, J., Meng, J., Sharma, U., Ma, J., Charboneau, R. and Roy, S. (2015). Morphine compromises bronchial epithelial TLR2/IL17R signaling crosstalk, necessary for lung IL17 homeostasis. Sci Rep 5: 11384.
  2. Cleaver, J. O., You, D., Michaud, D. R., Pruneda, F. A., Juarez, M. M., Zhang, J., Weill, P. M., Adachi, R., Gong, L., Moghaddam, S. J., Poynter, M. E., Tuvim, M. J. and Evans, S. E. (2014). Lung epithelial cells are essential effectors of inducible resistance to pneumonia. Mucosal Immunol 7(1): 78-88.
  3. Gay, N. J., Gangloff, M. and O’Neill, L. A. (2011). What the Myddosome structure tells us about the initiation of innate immunity. Trends Immunol 32(3): 104-9.
  4. Ho, P. C., Tsui, Y. C., Feng, X., Greaves, D. R. and Wei, L. N. (2012). NF-κB-mediated degradation of the coactivator RIP140 regulates inflammatory responses and contributes to endotoxin tolerance. Nat Immunol 13(4): 379-386.
  5. Xiong, Y., Qiu, F., Piao, W., Song, C., Wahl, L. M. and Medvedev, A. E. (2011). Endotoxin tolerance impairs IL-1 receptor-associated kinase (IRAK) 4 and TGF-β-activated kinase 1 activation, K63-linked polyubiquitination and assembly of IRAK1, TNF receptor-associated factor 6, and IκB kinase γ and increases A20 expression. J Biol Chem 286(10): 7905-7916.

简介

在这个协议中,我们描述近端连接测定(PLA),一种基于抗体的蛋白质相互作用检测方法。 这种方法依赖于个别一抗与两个推定的相互作用蛋白的特异性结合。 一抗需要有不同的宿主。 针对两种宿主的二抗具有与其连接的互补的寡核苷酸部分。 如果两种抗原紧密接近(推测彼此相互作用),则互补的寡核苷酸可以退火,并且荧光核苷酸可以并入单个DNA聚合步骤中。 在显微镜下,这些反应表现为点状荧光斑点,表明成功的PLA反应并提示两种抗原之间的蛋白质 - 蛋白质相互作用。

【背景】近端连接测定法(PLA)是基于抗体的技术,以确定两种蛋白质是否彼此具有40nm。以这种方式检测到的蛋白质可以通过荧光来识别(Ho等人,2012; Banerjee等人,2015)。这使得PLA成为定位蛋白质 - 蛋白质相互作用的极好工具。 Toll样受体(TLR)途径的激活是致病性威胁的先天性免疫应答的重要组成部分。 TLR识别病原体相关分子并诱导信号级联以实现对感染的快速响应。 TLR2和TLR4是TLR家族的两个研究得非常好的成员,它们对不同的刺激有反应。尽管两种受体都响应细菌感染而激活,但只有TLR4响应脂多糖暴露。它们激活一些共享的信号级联,但包括MyD88 / Traf6通路。该途径的诱导包括形成被称为myddosome的信号复合物(Gay等人,2011; Xiong等人,2011; Cleaver等人。,2014),一种蛋白质复合物,包括MyD88,IRAK1,IRAK4和Traf6等等。 Myddosome大会导致NF-κB介导的炎症反应和病原体清除。

可视化TLR信号通路的参与是鉴定和定位免疫应答的重要步骤。在这里,我们使用PLA来检测固定的肺组织和用LPS处理或暴露于机会性感染的培养的腹膜巨噬细胞系中的TLR通路活化。这种方法荧光标记相互作用,并保持在接近的蛋白质。使用荧光显微镜观察体内产生的标记使得我们能够鉴定组织位置方面的蛋白质复合物。在这里,我们证明了该试验能够在LPS处理腹膜巨噬细胞体外试验中检测机会性肺感染期间的TLR2活化和myddosome形成。我们还显示了该技术的特异性,因为它不表示LPS处理后的TLR2激活。

关键字:邻位连接分析, 蛋白质-蛋白质相互作用, Toll样受体, 荧光显微镜检查, 支气管上皮, 巨噬细胞, J774, Duolink

材料和试剂

  1. Nunc TM Lab-Tek TM II Chamber Slide TM系统(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:154534)
  2. 0.22μmPVDF针筒式过滤器(EMD Millipore,目录号:SLGV004SL)
  3. 盖玻片(22×40毫米)(VWR,目录号:470019-008)
  4. 从吗啡处理的和/或肺炎链球菌感染的小鼠收获的肺组织
  5. J774细胞,小鼠腹膜巨噬细胞系(ATCC,目录号:TIB-67)
  6. 一级抗体
    注意:用于PLA反应的每一对都需要通过蛋白质印迹(天然和SDS-PAGE)进行特异性验证,并且必须来自不同的宿主,与具有PLA探针的Duolink二抗相容。
    1. 亲和分离的兔抗TRAF6抗体(Sigma-Aldrich,目录号:SAB2102531)
      注:此产品已停产。
    2. 在兔中产生亲和力分离的抗TLR2抗体(Sigma-Aldrich,目录号:SAB1300199)
    3. 单克隆抗MYD88(克隆OTI2B2)(OriGene Technologies,目录号:TA502117)
  7. 连接到PLA探针的二抗
    1. Duolink原位PLA探针抗小鼠MINUS,亲和纯化的驴抗小鼠IgG(H + L)(Sigma-Aldrich,目录号:DUO92004)
    2. Duolink原位PLA探针抗兔PLUS,亲和纯化的驴抗兔IgG(H + L)(Sigma-Aldrich,目录号:DUO92002)
  8. 福尔马林溶液,中性缓冲液,10%(10%NBF)(Sigma-Aldrich,目录号:HT501128-4L)
  9. 石蜡(Fisher Scientific,目录号:P31-500)
  10. 二甲苯(组织学等级)(Fisher Scientific,目录号:X3S-4)
  11. 乙醇200标准(Merck,目录号:AX0441)
  12. 固定 - 透化缓冲液组(Thermo Fisher Scientific,eBiosciences TM,产品目录号:88-8824-00)
  13. pH 7.4的磷酸盐缓冲盐水(PBS)(1x)(Thermo Fisher Scientific,Gibco TM,产品目录号:10010023)
  14. Tween 20(Fisher Scientific,目录号:BP337-100)
  15. 用DAPI(Sigma-Aldrich,目录号:DUO82049)在Duolink原位固定培养基中进行培养。
  16. 指甲油(作为盖子密封胶)
  17. DMEM /高葡萄糖(4,500mg / L L-葡萄糖)(GE Healthcare,HyClone TM,目录号:SH30243.01)
  18. 青霉素 - 链霉素(10,000U / ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)

  19. 胎牛血清(FBS),合格的,USDA认可的区域(Thermo Fisher Scientific,Gibco TM,产品目录号:10437010)
  20. 超纯0.5M EDTA,pH6.0(Thermo Fisher Scientific,Invitrogen TM,目录号:15575020)
  21. 来自大肠杆菌O127:B8的脂多糖(Sigma-Aldrich,目录号:L3880)
  22. Duolink®原位洗涤缓冲液,荧光(Sigma-Aldrich,目录号:DUO82049)
  23. Duolink®原位检测试剂橙色(Sigma-Aldrich,目录号:DUO92007)
  24. Duolink原位洗涤缓冲液A和B(Sigma-Aldrich,目录号:DUO82047)
  25. Duolink 原位清洗缓冲液一种工作溶液(见食谱)
  26. Duolink 原位清洗缓冲液B工作溶液(见食谱)

设备

  1. 科普林罐(通用)
  2. 油标记(Aqua-hold pap pen)(Electron Microscopy Sciences,目录号:71311)
  3. 蔬菜蒸笼(通用)
  4. 滑动湿度室(Simport,型号:M920)
  5. 实验室摇床或摇杆
  6. 荧光过滤器(Leica)
    1. 滤波器49,激励G365,发射445/50
    2. 滤波器组43 HE,激励BP550 / 25,发射605/70
  7. 荧光显微镜

软件

  1. ImageJ软件( https://imagej.nih.gov/ij/

程序

  1. 抗体选择
    1. 为了鉴定蛋白质 - 蛋白质相互作用,必须使用识别两种蛋白质的一级抗体。由于抗体的特异性是至关重要的,因此建议在初始和变性条件下使用组织裂解物对抗体进行Western印迹分析。目标是在两种条件下获得最高的特异性,因为以下步骤可能在细胞内混合了初始和变性条件下的靶抗原。
    2. 一抗必须在来源物种中有所不同(例如,兔抗MyD88和小鼠抗TRAF6)。
    3. 每种寡核苷酸偶联的二抗都必须识别一种一抗。

  2. 样品制备
    1. 组织切片

      1. 在体内收集的组织应立即在10%NBF中固定,然后包埋入石蜡。
      2. 从石蜡块装载5-8微米的部分到幻灯片。
      3. 通过将载玻片浸入二甲苯浴(3-4次浸渍和15-20秒保存时间)并用连续的乙醇洗涤液(100,70,50,30和0%的水溶液)重新水化载玻片来脱蜡玻片。

      4. 加入透化缓冲液(1x)足以在4°C的黑暗中覆盖该部分40分钟
      5. 用PBS洗一次载玻片,并在植物蒸锅中孵育幻灯片1小时。
      注:在我们的例子中,我们使用从吗啡处理和/或肺炎球菌感染小鼠收获的肺组织。
    2. 培养的细胞载玻片
      1. 板上40,000到50,000细胞/毫升培养基,室幻灯片和孵育过夜,让细胞粘附到幻灯片浓度60-80%汇合。
      2. 对室内的细胞进行必要的处理。
      3. 去除媒体,并立即修复细胞,每个室加入10%的NBF,并在室温下孵育20分钟。
      4. 去除室壁。
      5. 用PBS洗三次幻灯片。
      6. 通过在室温下孵育载玻片与含0.01%吐温20的PBS 10分钟渗透。
      7. 用PBS洗三次幻灯片。
      注:在我们的例子中,我们使用小鼠腹腔巨噬细胞系J774。我们用不同浓度的LPS处理这些细胞1小时以诱导与TLR4结合的myddosome。

  3. PLA协议
    1. 轻拍幻灯片删除多余的液体,然后空气干燥,用油标记轮廓组织部分或井。

    2. 在湿度室中用封闭液孵育载玻片30分钟
    3. 去除阻断溶液,并添加足够的两种一抗重构在封闭液(大约1微克抗体/室或每节),以覆盖每个部分或室。
      注意:大约40μl的溶液将足以覆盖一个室。

    4. 在湿度室内孵育过夜,4°C
    5. 将幻灯片放在装有清洗缓冲液A的Coplin罐中(参见食谱),每次在实验室摇床上放置两次,每次5分钟。
    6. 加入寡核苷酸连接的第二抗体溶液,在洗涤缓冲液A(足以覆盖部分/细胞)中以1:10稀释至载玻片。

    7. 在湿度室中37°C孵育1小时
    8. 在洗涤缓冲液A中洗两次,每次五分钟在摇床上。
    9. 将连接酶溶液加入载玻片(足以覆盖切片/细胞),并在37℃的湿度室中孵育30分钟。
      注意:连接酶的工作浓度应为原瓶的1:40,用分子生物学级别的水稀释。
    10. 在洗涤缓冲液A中洗两次,每次五分钟在摇床上。
    11. 加入预混合的聚合酶和核苷酸溶液,在37℃孵育100分钟。
      注意:在这一点之后,限制幻灯片曝光。聚合酶的工作浓度应为原溶液的1:80,用分子生物学级水稀释。在洗涤缓冲液B(参见食谱)中洗涤两次,每次10分钟。
    12. 用1倍清洗缓冲液B清洗1分钟。
    13. 允许幻灯片干燥。
    14. 用DAPI在安装介质中安装盖玻片。
    15. 盖子边缘用指甲油打滑。
    16. 允许晾干。

数据分析

  1. 成像(图1)
    1. 使用荧光显微镜和适合DAPI的滤光片(如Leica滤光片组49),识别并拍摄幻灯片上的细胞/组织。

    2. 使用适合您的PLA探头的过滤器拍摄相同的区域 注意:
      1. 在我们的例子中,我们使用来自Leica的过滤器组43 HE来识别我们的PLA探针,Duolink原位检测试剂橙色。
      2. 除了合并的图像之外,请务必单独保存DAPI和PLA探针图像的单个图像文件。


        图1.使用ImageJ检测荧光颗粒如上所述使用颜色阈值。 A.DAPI染色的J774细胞的未编辑图像; B.应用色彩阈值后的相同图像; C.用于此阈值的设置的示例。

  2. 量化(图2)
    1. 确定细胞计数
      1. 在ImageJ中,打开包含DAPI图像的文件。
      2. 选择图片>调整>颜色阈值...
        注意:这适用于彩色图像。如果使用灰度图像,请选择图像>调整>阈值...
      3. 调整这个对话框中的参数,直到DAPI染色的细胞核完全,但明显突出。
      4. 选择分析>分析粒子...
      5. 选中“摘要”复选框,然后选择“确定”。
      6. 由此产生的“总结”窗口将包含核的总数。
    2. 确定PLA反应的次数
      1. 在Image J中,打开包含PLA图像的文件。
      2. 选择图片>调整>颜色阈值...
        注意:这适用于彩色图像。如果使用灰度图像,请选择图像>调整>阈值...
      3. 调整这个对话框中的参数,直到PLA反应点完全,但明显突出。
      4. 选择分析>分析粒子...
      5. 选中“摘要”复选框,然后选择“确定”。
      6. 由此产生的“总结”窗口将包含PLA反应的总数。

    3. 比较样品中PLA反应的数量与核的数量以确定每个细胞的蛋白质相互作用的数量 注意:这是一个简单的阳性PLA荧光和DAPI的比例
      1. 可以使用宏和批次分析来快速分析ImageJ中的多个图片。
      2. 如果图像中的细胞数量较低,手动计数可能更适合您的研究,并且更准确。


        图2.用于在荧光图像中为PLA反应创建颜色阈值的IJM语言宏的示例因为所使用的图像仅是PLA荧光而不是合并图像,因此最小和最大设置因为色调和饱和度可以是包含性的(第14,15行为色调,第17,18行为饱和度)。包含信号时调整最小亮度以消除背景(第20行)。宏创建一个蒙版,并在应用阈值后对剩余的粒子进行计数。通过使用ImageJ中内置的批处理功能,Macro可以用来一次对多个图像文件中的粒子进行计数。

  3. 结果
    1. J774细胞:J774是鼠巨噬细胞系,用不同浓度的LPS处理以激活TLR4途径。这需要将Myd88(作为更大的myddosome复合体的一部分)募集至TLR4,并且随后将其连接至Traf6(E3泛素连接酶)。如果TLR4被成功激活,Myd88和Traf6将在反应的第一个小时内结扎,如图3所示。


      图3.在J774细胞中显示MyD88 Traf6激活的近端连接分析在固定和PLA之前,用不同浓度的LPS处理细胞1小时。 A.未处理的J774细胞; B.2ng / ml LPS处理的J774细胞; C.定量每个DAPI染色的细胞的连接反应的数量。比例尺= 100微米。

    2. 肺切片:将C57b16 / j动物或者用吗啡或肺炎链球菌或者两者处理72小时,并且使用它们的肺切片在TLR2和Myd88之间进行PLA。如图4所示,单独的治疗没有表现出阳性的PLA反应。这是预期的,因为吗啡治疗本身不会激活TLRs,但会使个体易感染机会性感染。单独肺炎链球菌也不会激活TLR2,因为肺完整的上皮屏障。然而,联合治疗由于TLR2的激活而显示出阳性PLA反应。


      图4.显示小鼠肺上皮中的TLR2 / MyD88相互作用的PLA。 (A)72小时吗啡处理(B)用肺炎链球菌感染(C)72小时吗啡处理和<! - SIPO
    3. 阴性对照:除了每个实验内的单独对照之外,进行本实验以测试方法的完整性。如图5所示,我们对用LPS处理的J774细胞中的TLR2和Myd88进行了PLA反应。这个反应与预期的一样是阴性的,因为在这些条件下,LPS刺激TLR4和Myd88不应该与TLR2结合。


      图5.在用LPS处理的J774细胞中,PLA显示TLR2和MyD88之间没有相互作用我们在任何处理组中观察到很少或没有PLA反应。 LPS是TLR4的激活剂,而不是TLR2。尽管TLR2和MyD88抗体都可能与它们各自的抗原相互作用,但抗原并不存在于彼此的40nm内,并且不会诱导PLA反应。比例尺= 100微米。

    4. 阳性对照:我们验证了该方法检测蛋白质相互作用的能力。如图6所示,我们对用LPS处理的J774细胞中的TLR4和CD14进行了PLA反应。我们检测到TLR4和CD14之间的LPS依赖性相互作用。这是预期的,因为LPS处理下的TLR4 / CD14相互作用已经被彻底表征了。


      图6.显示用LPS处理30分钟的J774细胞中的TLR4 / CD14相互作用的近端连接分析。 :一种。未经处理的J774细胞; B. 0.4ng / ml LPS处理的J774细胞; C.2ng / ml LPS处理的J774细胞; D.每个DAPI染色的细胞的连接反应数目的量化。比例尺= 50微米。

笔记

如前所述,Duolink PLA方法在体外和体内条件下的重复性良好,并有注意事项。人们应该记住的考虑如下:

  1. 根据我们的经验,室内幻灯片的质量非常重要。尝试了各种品牌,Nunc品牌(上述材料中的第一)给了我们最清晰的结果。应避免使用合成基座的房间滑梯。质量好的光学透明玻璃基地是最好的。
  2. 我们已经使用8孔室幻灯片试剂优化,并有足够的实验重复(在这种情况下n = 4每个条件)。但是,在洗涤步骤中应该小心,因为这些腔室载玻片的边缘表面张力相当强,并且最佳的洗涤可能是具有挑战性的。在洗涤时,建议偶尔的旋转运动覆盖整个室,包括边缘。
  3. PLA最适合60-80%汇合细胞。我们一直喜欢这种浓度,因为它使腔室底部的贴片留空,这作为试剂的非特异性结合的附加控制。
  4. 对于体内实验,背景染色比在铺板细胞中更高。这是预期的,非特定的背景通常是一个统一的色调,而不是点状污点。由于在体内样品中观察到高水平的非特异性染色,您可能希望增加组织样品的阻断时间。这应当根据组织的来源来优化,例如与本文使用的肺组织相比,肠和脂肪组织可能需要更少或更多的孵育时间。
  5. 固定的组织载玻片必须在与单个抗体孵育之前进行抗原修复。
    流式细胞透化缓冲液和基于蔬菜蒸汽的抗原检索的组合一直给我们成功的PLA反应
  6. 通常,PLA反应总是产生点状荧光。在极少数情况下,如果PLA反应持续染色,应使用适当的实验对照和单一抗体对照来验证特异性反应。
  7. 在参考一级抗体的选择时,用于PLA反应的每一对需要通过蛋白质印迹(天然和SDS-PAGE)进行特异性验证,并且必须来自不同的宿主,与具有PLA探针的Duolink二抗相容。

食谱

  1. Duolink原位洗涤缓冲液A
    1. 另一种使用Duolink原位洗涤缓冲液A
      要制备1x缓冲液,将一个小袋的内容物溶解在高纯度水中,最终体积为1000 ml。
      在室温下储存袋子 注:到期日期在每个批次上标记。 1x溶液可以在室温下保存一小段时间(一周或更短)。长时间储存于4°C。使用前将溶液置于室温。
    2. 备选方案2 - 使您自己的Duolink原位洗涤缓冲液A

      1. 在800毫升高纯度水中溶解8.8克氯化钠,1.2克Tris碱和0.5毫升吐温20
      2. 使用HCl将pH调节至7.4
      3. 加入高纯度水至1,000 ml(最终浓度为0.01 M Tris,0.15 M NaCl和0.05%吐温20)

      4. 过滤溶液通过一个0.22微米的过滤器和存储在4°C
      5. 使用前请将溶液置于室温下
  2. Duolink原位洗涤缓冲液B
    1. 备用1用Duolink原位洗涤缓冲液B
      要制备1x缓冲液,将一个小袋的内容物溶解在高纯度水中,最终体积为1000 ml。
      在室温下储存袋子 注:到期日期在每个批次上标记。 1x溶液可以在室温下保存一小段时间(一周或更短)。长时间储存于4°C。使用前将溶液置于室温。
    2. 备选方案2 - 使您自己的Duolink原位洗涤缓冲液B

      1. 溶解5.84克NaCl,4.24克Tris碱和26.0克Tris-HCl在500毫升高纯度水中
      2. 使用HCl将pH值调节至7.5
      3. 将高纯度水加到1000毫升(终浓度为0.2M Tris和0.1M NaCl)

      4. 过滤溶液通过一个0.22微米的过滤器和存储在4°C
      5. 使用前请将溶液置于室温下

致谢

这项工作得到了NIH R21HL125021和迈阿密大学机构支持(SB)的部分支持。作者声明没有利益冲突。所有数据的数据都是专门为本协议生成的。然而,PLA方案适用于我们以前的出版物(Banerjee等人,2015),SciRep 5:11384的J774细胞;本协议的第一号参考文献),在16HBE14o(人支气管上皮细胞系)上进行。如文中所述,对肺组织的PLA进行了改良和修改。

参考

  1. Banerjee,S.,Ninkovic,J.,Meng,J.,Sharma,U.,Ma,J.,Charboneau,R.和Roy,S。(2015)。 吗啡妥协支气管上皮TLR2 / IL17R信号串扰,肺IL17稳态所必需 < em> Sci Rep 5:11384。
  2. Cleaver,JO,You,D.,Michaud,DR,Pruneda,FA,Juarez,MM,Zhang,J.,Weill,PM,Adachi,R.,Gong,L.,Moghaddam,SJ,Poynter,ME,Tuvim, MJ和Evans,SE(2014)。 肺上皮细胞是肺炎的诱导性抗性的重要效应物粘膜免疫学 7(1):78-88。
  3. Gay,N.J。,Gangloff,M.and O'Neill,L.A。(2011)。 Myddosome结构告诉我们什么是天然免疫的启动。 免疫学32(3):104-9。
  4. Ho,P.C.,Tsui,Y.C.,Feng,X.,Greaves,D.R。和Wei,L.N。(2012)。 NF-κB介导的共激活因子降解RIP140调节炎症反应并有助于耐受内毒素。 (Nat Immunol)13(4):379-386。
  5. Xiong,Y.,Qiu,F.,Piao,W.,Song,C.,Wahl,L.M。和Medvedev,A.E。(2011)。内毒素耐受能力损害IL-1受体相关激酶(IRAK)4和TGF-β激活的激酶(TGF-β1) 1激活,K63连接的多聚遍在蛋白化和IRAK1,TNF受体相关因子6和IκB激酶γ的装配,并增加A20表达。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mendez, R. and Banerjee, S. (2017). Proximal Ligation Assay (PLA) on Lung Tissue and Cultured Macrophages to Demonstrate Protein-protein Interaction. Bio-protocol 7(21): e2602. DOI: 10.21769/BioProtoc.2602.
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