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Oct 2019

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Immunofluorescent Staining of Claudin-2 in Cultured Kidney Tubular Cells
肾脏肾小管培养细胞中Claudin-2的免疫荧光染色   

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Abstract

Members of the claudin family of tight junction proteins regulate paracellular permeability and modulate cell signaling. During junction remodeling, these proteins are selectively inserted into or retrieved from the tight junctions, but the control and coordination of these processes remain incompletely understood. Visualization of claudins allows the assessment of changes in their localization and abundance. We use the described protocol to stain claudin-2, but it can also be adapted to stain any tight junction protein. We found that using methanol for fixing allows the best preservation of claudin-2 both at the membrane and in cytoplasmic vesicles. Staining is done using a claudin-2 specific primary and a fluorescently labelled secondary antibody, along with DAPI to label nuclei. The samples are then imaged using confocal microscopy, and a z-stack is obtained allowing visualization of both junctional and intracellular claudin-2. Total claudin-2 signal can be quantified after 3D reconstruction of the images using the Imaris software.

Keywords: Claudin (Claudin), Epithelial cells (上皮细胞), Confocal microscopy (共聚焦显微镜), Indirect immunofluorescent staining (免疫荧光间接染色), Fixed cells (固定细胞), Maximum intensity projection (最大密度投影), 3D reconstitution (三维重建), Analysis (分析)

Background

Tight junctions (TJ) are multiprotein complexes localized at the apical-most portion of the intercellular junctional complexes that connect epithelial cells (Van Itallie and Anderson, 2014). These structures generate a permeability barrier and ion-specific paracellular pathways, maintain apico-basal polarity, provide input for various vital functions and modulate signaling pathways. The proteins located at the TJs can be divided into transmembrane and associated cytoplasmic proteins (reviewed in (González-Mariscal et al., 2003). The transmembrane proteins can be further subdivided into three major categories: claudins, tight junction-associated Marvel proteins (e.g., occludin) and single span proteins (e.g., Junctional Adhesion Molecules). TJ-associated cytosolic proteins include a large array of adaptors connected to signaling and cytoskeletal proteins. Collectively, these proteins are referred to as the cytoplasmic plaque. Claudins are small molecular weight (20-34 kDa) tetraspan membrane proteins that are essential components of the TJs (Tsukita et al., 2019). They incorporate into the TJ strands that typically contain a mosaic of various claudin isoforms that determine permselectivity (Van Itallie and Anderson, 2004). Interestingly, claudins have also recently emerged as key modulators of signaling, an effect that is likely due to the interactions with cytosolic adapters.

In this protocol we focus on claudin-2 (Cldn-2), a 230 amino acid member of the family, with a calculated molecular mass of 24.5 kDa. Cldn-2 was first described by Dr Shoichiro Tsukita and coworkers (Furuse et al., 1998a and 1998b) (for review see Venugopal et al., 2019). It is highly enriched in the kidney proximal tubules (Enck et al., 2001) and in intestinal and liver cells (e.g., Sakaguchi et al., 2002; Escaffit et al., 2005). It generates a cation selective paracellular channel, and therefore, its presence corresponds to elevated paracellular permeability (e.g., Amasheh et al., 2002). Multiple studies have revealed that in various cells Cldn-2 can be found not only at the membrane, i.e., in the TJs, but also in intracellular vesicles (Dukes et al., 2012; Lu et al., 2014; Amoozadeh et al., 2015), and in the nucleus (Ikari et al., 2014; Amoozadeh et al., 2018).

Many studies have used immunofluorescent staining in fixed cells followed by confocal microscopy to visualize TJ proteins and analyze their localization and abundance. These approaches allow investigation of the mechanisms that regulate TJ protein trafficking. The protocols differ in the fixation and blocking method and use primary antibodies targeted against specific proteins. Here we describe the method used in our lab to stain Cldn-2 in cultured kidney tubular cells based on (Amoozadeh et al., 2015 and 2018; Dan et al., 2019). We also detail our protocol for 3D reconstitution and analysis of changes in staining intensity following treatment, e.g. with cytokines. While we focus specifically on claudin-2, the same approach can be used for other TJ proteins in any epithelial or endothelial cells.

Materials and Reagents

  1. Micro cover glasses, round, No. 1, 18 mm (VWR® International, catalog number: CA-48380-046 ), sterilize coverglasses using autoclave
  2. Microscope slides (Fisherbrand, Fisher Scientific, catalog number: 12-550-15 )
  3. Clear tissue culture-treated 12-well microplates, (Corning Costar®, Millipore-Sigma, catalog number: CLS3513 )
  4. Parafilm
  5. Paper towel
  6. Cell line: LLC-PK1 kidney tubule epithelial cell line (European Collection of Authenticated Cell Cultures (Salisbury, UK), catalog number: 86121112 )
  7. For cell culture:
    1. Dulbecco's Modified Eagle Medium (D-MEM), low glucose, with L-glutamine
    2. 110 mg/L sodium pyruvate (Thermo Fisher, Gibco, catalog number: 11885084 )
    3. 10% Fetal Bovine Serum (FBS) (Thermo Fisher, Gibco, catalog number: 12483-020 )
    4. 1% Penicillin-Streptomycin (Penicillin-Streptomycin, 100x, sterile-filtered, cell culture tested) (Millipore-Sigma Aldrich, catalog number: P-4333 )
    5. Trypsin-EDTA (0.05% Trypsin with EDTA 4Na) (Thermo Fisher, Gibco, catalog number: 25300062 )
    6. Phosphate buffered saline (PBS) (Thermo Fisher, Gibco, catalog number: 10010023 )
  8. Methanol (BioShop Canada, catalog number: Met302 ), cool to -20 °C before using
  9. 10x PBS Buffer (pH 7.4 Sterile, w/o Ca, Mg) (BioShop Canada, catalog number: PBS415 ), open in hood to maintain sterility for storage
  10. Bovine serum albumin (BSA) (fraction V, heat shock isolation, > 98%) (BioShop Canada, catalog number: ALB001 )
  11. Polyclonal rabbit anti-Claudin-2 antibody (Thermo Fisher Scientific, Invitrogen, catalog number: 28530 ), aliquot to avoid repeated thawing, store at -20 °C
  12. Fluorophore-labeled secondary antibody:
    Donkey anti-Rabbit IgG labelled with Alexa Fluor 488 (green) (Thermo Fischer Scientific, catalog number: A-21206 )
    or
    Donkey anti-Rabbit IgG labelled with Alexa Fluor 555 (red) (Thermo Fischer Scientific, catalog number: A- A31572 )
  13. 2,4-diamidino-2-phenylindole (DAPI) (Thermo Fischer Scientific, catalog number: D1306 )
    Make a 5 mg/ml DAPI stock solution by dissolving the contents of one vial (10 mg) in 2 ml of deionized water. Aliquot the stock, protect from light. DAPI stock solution can be stored at 4 °C for up to 6 months, or at -20 °C for longer periods.
  14. Fluorescent mounting medium (Dako, Agilent catalog number: S3023 ), store at 4 °C
  15. Cell culture medium (see Recipes)
  16. 1x PBS (see Recipes)
  17. Blocking buffer (see Recipes)
  18. Antibody buffer (see Recipes)

Equipment

  1. Tweezers
  2. Nikon Eclipse TS100 microscope
  3. WaveFX spinning-disk confocal microscope (Quorum Technologies, Guelph, Canada) with an ORCA-flash4.0 digital camera with Gen II sCMOS image sensor

Software

  1. Metamorph Image Analysis Software, Molecular Devices
  2. Imaris software 8.0.2 (Bitplane)

Procedure

  1. Seeding cells on cover glasses
    1. Grow LLC-PK1 cells in a T75 flask with culture medium (low glucose DMEM supplemented with 10 % FBS and 1% Pen-Strep, see Recipes) in an incubator with 5% CO2. Use flask of LLC-PK1 cells grown to about 90% confluence. Rinse with 5-8 ml PBS and add 1-2 ml trypsin according to standard cell culture procedures. Place back into incubator for about 10 min, until cells are floating. Note that details of culturing have to be adjusted for specific cell lines.
    2. While cells are in trypsin, place autoclaved coverglass in multi-well plates using sterile tweezers.
    3. When cells are floating, resuspend them in 8 ml fresh medium, mix well and seed an equal number of cells onto coverglasses. Use 2 x 105-5 x 105/ml cells in 1 ml medium/well (12-well plate).
    4. Gently shake the culture dish to evenly distribute the cells and place into culture incubator.
    5. Grow cells to 100% confluency (about 48 h). Of note, we found that expression of Cldn-2 is low in subconfluent cell cultures and increases as the cells become confluent (Amoozadeh et al., 2018). Therefore, it is important to allow the cells to become fully confluent and generate mature TJs, before performing the immunostaining (see Figure 1).


      Figure 1. Subconfluent (A) and confluent (B) LLC-PK1 cultures. Phase contrast images of live cells grown on cover glasses were obtained using a Nikon eclipse TS100 microscope (10x objective).

  2. Fixation
    1. Treat cells according to experimental needs, e.g., with cytokines. At the end of the treatment, aspirate the medium and quickly rinse with 1x PBS. The steps after rinsing do not need to be performed under sterile conditions. The multi-well dish used for culturing cells can be used for the staining protocol under non-sterile conditions.
    2. Aspirate PBS and immediately fix with 0.5-1 ml methanol (stored at -20 °C) for 5 min. Make sure the methanol fully covers the cover glasses. These 2 steps should be done as fast as possible.
    3. Wash with 1x PBS 5 times. After this step the samples can be stored overnight at 4 °C if needed. However, we prefer to proceed to the next steps immediately. Longer storage is not recommended.

  3. Blocking, staining and mounting
    1. Block with 0.5-1 ml blocking buffer in PBS for 1 h at room temperature. Make sure the buffer fully covers the cover glasses.
    2. Primary antibody incubation:
      1. Dilute antibody 1:100 in antibody buffer.
      2. To reduce antibody use, place 50 μl of diluted antibody on parafilm and place the cover glass with cells facing the drop. Avoid bubbles and don’t let cover glass dry out. Incubate for 1h at room temperature.
      3. Non-specific secondary antibody binding control: use one of the cover glasses to demonstrate specificity of the secondary antibody. Omit primary antibody but do all other steps similarly.
      4. Control sample for primary antibody specificity: use samples where Cldn-2 was silenced or knocked down (Dan et al., 2019).
    3. At the end of the incubation use tweezers to place cover glasses back into the same culture dish turning the cells upside. Wash with 1x PBS 5 times.
    4. Secondary antibody incubation:
      1. Dilute secondary antibody (1:1,000) and DAPI (1:5,000) in antibody buffer.
      2. Add 500 μl antibody solution to the cover glasses.
      3. Incubate for 1h at room temperature in the dark.
    5. Wash with 1x PBS 5 times over 15 min.
    6. Label microscope slides to identify samples. Place a drop of fluorescent mounting medium on a microscope slide. Lift up cover glasses using a tweezer and touch the edge to a paper towel to remove excess fluid. Place stained cover glass onto the drop of fluorescent mounting medium with cells facing down. Avoid air bubbles. Aspirate access fluid.
    7. Let the fluorescent mounting medium dry for at least 30 min before imaging or storing. Store dried slides in the dark at 4 °C.

  4. Visualization using confocal microscopy
    1. Set up the confocal microscope
      1. Settings (e.g., laser, objective etc) will vary depending on your experiment. We use a 63x objective, which was chosen to give the desired resolution and size of the imaged field.
      2. Chose the excitation/emission settings for the fluorophore of your secondary antibody (Alexa488 or 555 in our experiments) and DAPI.
      3. Select exposure time to avoid image saturation. Obtain z-stack sections at both wavelength through the whole sample by defining the top and bottom focal plane positions and a step size of around 2 μm (12-16 focal planes).
    2. A single plane at the level of the TJs of the stack can be used for display. However, LLC-PK1 cells in a monolayer often have slight height differences and can also form domes. This causes the TJs in different areas of a visual field to be at slightly different focal planes, making some areas to be out of focus. Further, TJ-localized and intracellular Cldn-2 are also in different focal planes. To visualize all Cldn-2 within the cells, use Metamorph to generate a Maximum Intensity Projection from the z-stack (see Figure 2 for an example). This process uses the brightest pixel from each layer and displays its intensity value in the final image, thus allowing visualization of a 3D image collapsed into a 2D picture. The processing must be disclosed in the manuscript.


      Figure 2. Typical Claudin-2 staining in LLC-PK1 cells. LLC-PK1 cells were fixed with methanol, and Cldn-2 stained as described in the protocol. The nuclei were counterstained with DAPI. Z-stacks were obtained using confocal microscopy for each staining, maximal intensity projection pictures were generated using the Metamorph software. The images obtained for the two wavelengths were combined. The bar represents 10 μm.

Data analysis

Image processing and data analysis:

  1. 3D reconstruction In Imaris
    To quantify intensity of total Cldn-2 staining, we use the Imaris software for a 3D reconstruction that can then be used for quantification. Of note, the above generated Maximum Intensity Projection images cannot be used for quantification. For basic familiarity with the Imaris software, users are referred to free online tutorials at http://www.bitplane.com/learning.
    1. Upload z-stack images to the Imaris 8.0.2 software.
    2. Click ‘Edit/Add Channels’ and select the appropriate image.
    3. In the main menu, Select “Surpass/Add new surfaces” and click ‘Create Surface’.
    4. Select the appropriate ‘Surface Area Detail Level’. Select ‘Absolute Intensity’ for thresholding.
      Note: Both settings are automatically calculated, however, users can alter these parameters. Keep these settings constant throughout image analysis.
    5. Click ‘Finish’ to complete 3D reconstruction of the stack.
    6. Create a 3D spot object for quantifying the number of nuclei by adding a new spot object.
    7. Select ‘Different Spot Size’.
    8. For ‘Source Channel’, select the DAPI channel.
    9. For ‘Spot Detection’, enter ‘Estimated XY Diameter and Z Diameter’ of the nuclei.
    10. Manually adjust the ‘Spot Quality’ to include any undetected nuclei.
    11. Select ‘Intensity Max = masked channel number of the cell outline’ to restrict extra nuclei spots. Note that the values for all the described parameters depend on the properties of the images acquired and will differ in specific images.
    12. Adjust ‘Spot Region Threshold’ and ensure all nuclei spots are detected.
    13. Click ‘Finish’ to complete the detection of nuclei spots.
    14. Select the required statistic parameters (volume and total number of spots) and export the .csv files. These can be opened using a graph program.

  2. Data analysis
    1. Use a graph program (Excel or GraphPad) to calculate the sum of volumes of each surface (μm3).
    2. Express the data as total surface volume per cell (nuclei spots).

Notes

  1. Cell line choice: The LLC-PK1 cell line we use originated from the European Collection of Authenticated Cell Cultures. This LLC-PK1 clone was originally also available at ATCC (ATCC® CRL-1392TM) but has since been discontinued. ATCC distributes another LLC-PK1 cell line, however, we found that LLC-PK1 cells from various sources differ in their claudin-2 expression levels. Thus, prior to choosing a cell line for the project, good expression of the claudin of interest should be verified.
  2. Cell counting and confluency: TJs develop as the layer becomes confluent and therefore it is recommended that fully confluent layers are used. Claudin-2 total expression is low in subconfluent cells and increases as confluency develops. Confluency should be kept constant when comparing different conditions. We found that when the same conditions are consistently applied during culture and seeding, cell numbers can be kept constant. Thus, after establishing the protocol, routine cell counting is not necessary. However, experimental manipulations might alter cell growth and thus confluency should be routinely checked and considered as an important variable.
  3. Throughout the staining process, drying of the cover glasses must be avoided.
  4. The protocol described here is used to stain claudin-2. The same protocol can be used to visualize other junction proteins in any epithelial cell lines. We found that the mode of fixation is key for good preservation and visualization of junctional proteins. We had good results using methanol when staining claudins. For occludin we used acetone fixation. For these fixation methods additional permeabilization is not needed. For a detailed analysis of the various fixation methods used for junctional proteins, please refer to Buckley et al., 2018.
  5. The quantification described is not specific for TJ-localized claudin-2 but reflects total claudin-2 abundance, including cytosolic protein. In our experiments the quantification was used for a comparison between control and TNF-α-treated samples. The data were expressed as staining/cell, relative to the control (untreated) to demonstrate fold changes. In this case, the control, untreated samples serve as reference. For other type of comparisons (e.g., comparison of untreated samples), a reference protein, e.g., another TJ protein should be used (e.g., costaining for occludin or ZO-1 and expressing ratio of claudin-2 to occludin staining). The choice of the reference however can pose difficulties for cytokine-treated samples, as we found that expression of all TJ proteins we tested (e.g., other claudins, occludin), as well as the adherens junction protein E-cadherin were all altered by TNFα (Amoozadeh et al., 2015). Thus, before choosing a reference, it is important to show that it is unaltered by the treatment. Finally, quantification should be verified by other quantitative methods, such as Western blotting.

Recipes

  1. Cell culture media
    Dulbecco's Modified Eagle Medium (D-MEM), low glucose, with L-glutamine and 110 mg/L sodium pyruvate was supplemented with 10% Fetal Bovine Serum (FBS) and sterile-filtered 1% Penicillin-Streptomycin
  2. 1x PBS
    Dilute from 10x PBS: add 50 ml 10x PBS to 450 ml distilled H2O
  3. Blocking buffer
    3% BSA in PBS
    1. Mix 1.5 g BSA in 50 ml PBS
    2. Store short term (1 day) at 4 °C
    Long-term storage not recommended
  4. Antibody buffer
    0.3% BSA in PBS
    1. Mix 0.15 g BSA in 50 ml PBS
    2. Store short term (1 day) at 4 °C
    Long-term storage not recommended

Acknowledgments

Funding: Kidney Foundation of Canada, Canadian Institutes of Health Research (CIHR) grants PJT-149058 and MOP-142409.
   This protocol was derived from: Amoozadeh et al., 2015 and 2018; Dan et al., 2019.

Competing interests

The authors declare that they have no financial or non-financial conflicts of interest.

References

  1. Amasheh, S., Meiri, N., Gitter, A. H., Schoneberg, T., Mankertz, J., Schulzke, J. D. and Fromm, M. (2002). Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells. J Cell Sci 115(Pt 24): 4969-4976.
  2. Amoozadeh, Y., Anwer, S., Dan, Q., Venugopal, S., Shi, Y., Branchard, E., Liedtke, E., Ailenberg, M., Rotstein, O. D., Kapus, A. and Szaszi, K. (2018). Cell confluence regulates claudin-2 expression: possible role for ZO-1 and Rac. Am J Physiol Cell Physiol 314(3): C366-C378.
  3. Amoozadeh, Y., Dan, Q., Xiao, J., Waheed, F. and Szaszi, K. (2015). Tumor necrosis factor-α induces a biphasic change in claudin-2 expression in tubular epithelial cells: role in barrier functions. Am J Physiol Cell Physiol 309(1): C38-50.
  4. Buckley, A. G., Looi, K., Iosifidis, T., Ling, K. M., Sutanto, E. N., Martinovich, K. M., Kicic-Starcevich, E., Garratt, L. W., Shaw, N. C., Lannigan, F. J., Larcombe, A. N., Zosky, G., Knight, D. A., Rigby, P. J., Kicic, A. and Stick, S. M. (2018). Visualisation of multiple tight junctional complexes in human airway epithelial cells. Biol Proced Online 20: 3. 
  5. Dan, Q., Shi, Y., Rabani, R., Venugopal, S., Xiao, J., Anwer, S., Ding, M., Speight, P., Pan, W., Alexander, R. T., Kapus, A. and Szászi, K. (2019). Claudin-2 suppresses GEF-H1, RHOA, and MRTF, thereby impacting proliferation and profibrotic phenotype of tubular cells. J Biol Chem 294(42): 15446-15465.
  6. Dukes, J. D., Whitley, P. and Chalmers, A. D. (2012). The PIKfyve inhibitor YM201636 blocks the continuous recycling of the tight junction proteins claudin-1 and claudin-2 in MDCK cells. PLoS One 7(3): e28659.
  7. Enck, A. H., Berger, U. V. and Yu, A. S. L. (2001). Claudin-2 is selectively expressed in proximal nephron in mouse kidney. Am J Physiol Renal Physiol 281(5): F966-F974.
  8. Escaffit, F., Boudreau, F. and Beaulieu, J. F. (2005). Differential expression of claudin-2 along the human intestine: Implication of GATA-4 in the maintenance of claudin-2 in differentiating cells. J Cell Physiol 203(1): 15-26.
  9. Furuse, M., Fujita, K., Hiiragi, T., Fujimoto, K. and Tsukita, S. (1998a). Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141(7): 1539-1550.
  10. Furuse, M., Sasaki, H., Fujimoto, K. and Tsukita, S. (1998b). A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts. J Cell Biol 143(2): 391-401.
  11. González-Mariscal, L., Betanzos, A., Nava, P. and Jaramillo, B. E. (2003). Tight junction proteins. Prog Biophys Mol Biol 81(1): 1-44.
  12. Ikari, A., Watanabe, R., Sato, T., Taga, S., Shimobaba, S., Yamaguchi, M., Yamazaki, Y., Endo, S., Matsunaga, T. and Sugatani, J. (2014). Nuclear distribution of claudin-2 increases cell proliferation in human lung adenocarcinoma cells. Biochim Biophys Acta 1843(9): 2079-2088.
  13. Lu, R., Johnson, D. L., Stewart, L., Waite, K., Elliott, D. and Wilson, J. M. (2014). Rab14 regulation of claudin-2 trafficking modulates epithelial permeability and lumen morphogenesis. Mol Biol Cell 25(11): 1744-1754.
  14. Sakaguchi, T., Gu, X., Golden, H. M., Suh, E., Rhoads, D. B. and Reinecker, H. C. (2002). Cloning of the human claudin-2 5'-flanking region revealed a TATA-less promoter with conserved binding sites in mouse and human for caudal-related homeodomain proteins and hepatocyte nuclear factor-1α. J Biol Chem 277(24): 21361-21370.
  15. Tsukita, S., Tanaka, H. and Tamura, A. (2019). The claudins: from tight junctions to biological systems. Trends Biochem Sci 44(2): 141-152.
  16. Van Itallie, C. M. and Anderson, J. M. (2004). The molecular physiology of tight junction pores. Physiology (Bethesda) 19: 331-338.
  17. Van Itallie, C. M. and Anderson, J. M. (2014). Architecture of tight junctions and principles of molecular composition. Semin Cell Dev Biol 36: 157-165.
  18. Venugopal, S., Anwer, S. and Szaszi, K. (2019). Claudin-2: roles beyond permeability functions. Int J Mol Sci 20(22). Doi.org/10.3390/ijms20225655.


简介

[摘要 t] 紧密连接蛋白claudin家族的成员调节细胞旁通透性并调节细胞信号传导。在连接重塑过程中,这些蛋白被选择性地插入紧密连接或从紧密连接中检索出来,但是对这些过程的控制和协调仍不完全了解。claudins的可视化可以评估其定位和丰度的变化。我们使用所描述的方案对claudin-2染色,但它也可以用于染色任何紧密连接蛋白。我们发现使用甲醇进行固定可以使claudin-2在膜和细胞质囊泡中得到最佳保存。使用claudin-2特异性一抗和荧光标记的二抗以及与DAPI标记核一起进行染色。然后使用共聚焦显微镜对样品成像,并获得z堆栈,从而可以可视化连接和细胞内claudin-2。总的claudin-2信号可以在3D重建图像后使用Imaris 软件。

[背景 ] 紧密连接(TJ)是一种多蛋白复合物,位于连接上皮细胞的细胞间连接复合物的最顶端(Van Itallie和Anderson,2014)。这些结构产生通透性屏障和离子特异性旁细胞途径,维持apicobasal 极性,为各种重要功能提供输入并调节信号传导途径。位于TJs的蛋白可分为跨膜蛋白和相关的胞质蛋白(综述见(González-Mariscal 等,2003)。跨膜蛋白可进一步分为三大类:claudins,紧密连接相关的Marvel蛋白(例如,闭合蛋白)和单跨度蛋白(例如,连接粘附分子).TJ相关胞质蛋白包括一个大的阵列连接到信令和细胞骨架蛋白适配器。总的来说,这些蛋白被称为胞质斑块。密蛋白皆s 商场分子量(2 0 - 34 kDa的)tetraspan 膜蛋白属于紧密连接的基本组分(Tsukita 。等人,2019) 。它们纳入TJ链通常包含确定各种密蛋白同种型的马赛克选择渗透性(范Itallie和Anderson,2004年。)有趣的是,claudins最近也已成为信号转导的关键调节剂,这种作用可能是由于相互作用引起的。与胞质适配器。

在此协议中,我们专注于claudin-2(Cldn-2),该家族的230个氨基酸成员,计算的分子量为24.5 kDa 。CLDN-2首先由医生描述昭一郎Tsukita 和同事(古濑等人,1998年和1998年)(对于综述参阅Venugopal 等人,2019 )。其高度富集于肾近端小管(Enck 等,2001)以及肠和肝细胞(例如,Sakaguchi 等,2002 ;Escaffit 等,2005)。它产生阳离子选择性的细胞旁通道,因此,其存在对应于细胞旁通透性升高(例如,Amasheh 等,2002 )。多项研究表明,不仅在膜上,即在TJs中,而且在细胞内囊泡中,都可以在各种Cldn-2细胞中发现细胞(Dukes 等人,2012; Lu 等人,2014; Amoozadeh 等人。,2015)和细胞核中(Ikari 等,2014; Amoozadeh 等,2018)。

许多研究使用固定细胞中的免疫荧光染色,然后通过共聚焦显微镜观察TJ蛋白并分析其定位和丰度。这些方法允许研究调节TJ蛋白运输的机制。方案在固定和封闭方法上有所不同,并使用针对特定蛋白质的一抗。在这里,我们基于(Amoozadeh 等人,2015和2018; Dan 等人,2019),介绍了我们实验室在培养的肾小管细胞中对Cldn-2染色的方法。我们还详细介绍了3D重建和分析处理后(例如使用细胞因子)染色强度变化的方案。尽管我们专门研究claudin-2,但相同的方法可用于任何上皮或内皮细胞中的其他TJ蛋白。

关键字:Claudin, 上皮细胞, 共聚焦显微镜, 免疫荧光间接染色, 固定细胞, 最大密度投影, 三维重建, 分析

材料和试剂


 


1. 微Ç 超过克lasses,- [R ound,没有。1 ,18毫米(VWR ® 国际,目录号:CA-48380-046),小号teriliz È 盖玻片使用高压釜       


2. 显微镜载玻片(Fisherbrand ,Fisher Scientific,目录号:12-550-15)       


3. 清除组织培养处理的12孔微量培养板(Costar公司康宁® ,Millipore公司-Σ,C atalog号:CLS3513)       


4. 封口膜       


5. 纸巾       


6. 细胞系:LLC-PK 1 肾小管上皮细胞系(欧洲认证细胞培养物保藏中心(英国索尔兹伯里),目录号:86121112)      


7. 对于细胞培养e:      


Dulbecco改良的Eagle培养基(D-MEM),低糖,含L-谷氨酰胺
110 mg / L 丙酮酸钠(Thermo Fisher ,Gibco,目录号:11885084)
10%胎牛血清(FBS)(Thermo Fisher,Gibco,目录号:12483-020)
1%青霉素-链霉素(青霉素-链霉素,100 x ,无菌过滤,经过细胞培养测试)(Millipore-Sigma Aldrich,目录号: P-4333)
胰蛋白酶-EDTA(0.05%胰蛋白酶与EDTA 4Na)(Thermo Fisher,Gibco,目录号:25300062)
磷酸盐缓冲盐水(PBS)(Thermo Fisher,Gibco,目录号:10010023)
8. 蛋氨酸hanol(BioShop 加拿大,目录号:Met302),Ç OOL至-20 ° 下用前       


9. 10X PBS缓冲液(pH 7.4的无菌,W / O型的Ca,Mg)的(BioShop 加拿大,目录号:PBS415)ö 笔在罩保持用于存储无菌       


10. 牛血清白蛋白(BSA)(馏分V,热休克隔离度,> 98%)(加拿大BioShop ,目录号:ALB001)   


11. 多克隆兔抗紧密连接蛋白-2抗体(赛默飞世尔科技,Invitrogen,目录号:28530),一个liquot以避免反复冻融,储存在-20    °C


12. 荧光团标记的二抗:   


用Alexa Fluor 488标记的驴抗兔IgG(绿色)(Thermo Fischer Scientific,目录号:A-21206)


要么


用Alexa Fluor 555(红色)标记的驴抗兔IgG(Thermo Fischer Scientific,目录号:A-A31572)


13. 2,4-二mid基-2-苯基吲哚(DAPI)(Thermo Fischer Scientific ,目录号:D1306)   


通过将一个小瓶(10 mg)的内容物溶解在2 ml去离子水中制成5 mg / ml DAPI储备溶液。分装库存,避光。DAPI储备溶液可以在4 °C下保存长达6个月,或在-20°C下保存更长时间。


14. 荧光封固介质(Dako公司,安捷伦目录号:S3023),小号撕在4℃下   


15. 细胞培养基(请参阅食谱)   


16. 1x PBS(请参阅食谱)   


17. 阻塞缓冲区(请参见食谱)   


18. 抗体缓冲液(请参阅食谱)   


 


设备


 


镊子
尼康E Clipse TS100显微镜
WaveFX 旋转盘共聚焦显微镜(Quorum Technologies,加拿大圭尔夫),带有带有第二代sCMOS 图像传感器的ORCA-flash4.0数码相机
 


软件


 


变形图像分析软件,分子设备
Imaris 软件8.0.2(Bitplane )
 


程序


 


盖玻片上接种细胞
生长LLC-PK 1个细胞在培养基中的T75烧瓶(低葡萄糖DMEM补充有10%FBS和1%青霉素-链霉素,见ř ecipes)在含5%CO的培养箱2 。使用生长至约90%汇合的LLC-PK 1 细胞烧瓶。根据标准细胞培养程序,用5-8 ml PBS 冲洗并添加1-2 ml 胰蛋白酶。放回培养箱约10分钟,直到细胞漂浮。需要注意的是培养的细节必须为特定的细胞系进行调整。
当细胞处于胰蛋白酶中时,使用无菌将高压灭菌的盖玻片置于多孔板中镊子。
当细胞漂浮时,将其重悬于8 ml 新鲜培养基中,混合均匀,将等量的细胞接种到盖玻片上。在1 ml培养基/孔(12孔板)中使用2 x 10 5 -5 x 10 5 / ml细胞。
轻轻摇动培养皿,使细胞均匀分布并放入培养箱中。
使细胞生长至100%融合(约48小时)。值得注意的是,我们发现亚融合细胞培养物中Cldn-2的表达较低,并随着细胞融合而增加(Amoozadeh et al。,2018)。因此,在进行免疫染色之前,使细胞完全融合并产生成熟的TJ非常重要(请参见图1)。
 


D:\重新格式化\ 2020-4-7 \ 1903016--1411 KatalinSzászi832528 \图jpg \图1.jpg


图1. š ubconflue NT (甲)和汇合(乙)LLC-PK 1个培养物。使用Nikon eclipse TS100显微镜(10倍物镜)获得在盖玻片上生长的活细胞的相差图像。


 


固定
根据实验的需要,治疗的细胞例如,用细胞因子。在治疗结束时,吸出培养基并用1x PBS快速冲洗。漂洗后的步骤无需在无菌条件下执行。用于培养细胞的多孔皿可以在非无菌条件下用于染色方案。
立即用0.5-1 m L 甲醇(储存在-20°C)固定PBS和抽吸液5分钟。确保甲醇完全覆盖了防护玻璃。这两个步骤应尽快完成。
用1x PBS洗涤5次。完成此步骤后,如果需要,可以将样品在4°C下保存过夜。但是,我们希望立即进行后续步骤。不建议更长的存储时间。
 


阻塞,染色和安装
用0.5块- 1米升的PBS封闭缓冲液在室温下1个小时。确保缓冲液完全覆盖了防护玻璃。
一抗孵育:
在抗体缓冲液中稀释抗体1:100。
为了减少抗体的使用,将50μl 稀释的抗体放在封口膜上,并将盖玻片的细胞面向液滴。避免产生气泡,也不要让玻璃盖变干。在室温下孵育1小时。
非特异性二抗结合控制:使用其中一个盖玻片证明二抗的特异性。省略一抗,但类似地执行所有其他步骤。
一抗特异性的对照样品:使用Cld n -2被沉默或敲低的样品(Dan 等,2019)。
孵育结束后,用镊子将盖玻片放回同一培养皿中,使细胞向上。用1x PBS洗涤5次。
二级抗体孵育:
在抗体缓冲液中稀释二抗(1:1,000)和DAPI(1:5,000)。
加入500 微升的抗体溶液到盖玻璃。
在黑暗中于室温下孵育1小时。
在15分钟内用1x PBS洗涤5次。
标记显微镜载玻片以识别样品。将一滴荧光固定剂放在显微镜载玻片上。使用镊子提起防护玻璃罩,将边缘碰到纸巾上,以去除多余的液体。将沾有污渍的盖玻片放在荧光固定介质滴上,并使细胞朝下。避免气泡。吸入流体。
在成像或存放之前,让荧光固定剂干燥至少30分钟。将干燥的载玻片在4°C下黑暗保存
 


使用共聚焦显微镜可视化
设置共聚焦显微镜
设置(例如,激光,目标等)将根据您的实验而异。我们使用63倍物镜,其目的是为了提供所需的分辨率和成像场的大小。
选择第二抗体(在我们的实验中为Alexa488或555)和DAPI的荧光团的激发/发射设置。
选择曝光时间以避免图像饱和。通过定义顶部和底部焦平面位置以及大约2μm (12-16个焦平面)的步长,在整个样本的两个波长下获得z堆栈截面。
可以使用堆栈TJ级别的单个平面进行显示。但是,单层中的LLC-PK 1 细胞通常具有微小的高度差异,也可以形成圆顶。这会导致视野不同区域中的TJ位于稍微不同的焦平面上,从而使某些区域失去焦点。此外,TJ 定位的和细胞内的Cldn-2也位于不同的焦平面中。要可视化细胞内的所有Cldn-2,请使用Metamorph 从z堆栈生成最大强度投影(示例请参见图2)。Ť ħ 是亲塞斯使用最亮像素从每个层,并显示其在最终图像中的强度值,从而允许折叠成2D画面的3D图像的可视化。处理过程必须在手稿中披露。
C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ 2020-7-20 \ 3678--1903016--1411 KatalinSzászi832528 \ Figs jpg \ Fig 2.jpg


图2. LLC-PK 1 细胞中典型的Claudin-2染色。将LLC-PK 1 细胞用甲醇固定,并按照方案中所述对Cldn-2进行染色。用DAPI 对细胞核染色。使用共聚焦显微镜对每种染色获得Z轴,使用Metamorph 软件生成最大强度的投影图像。将针对两个波长获得的图像进行合并。条形代表10μm 。


 


数据分析


 


图像p rocessing和d ATA 一个nalysis :


Imaris的3D重建
为了量化总Cldn-2染色的强度,我们使用Imaris 软件进行3D重建,然后将其用于量化。值得注意的是,以上生成的最大强度投影图像无法用于量化。为了基本了解Imaris 软件,请访问http://www.bitplane.com/learning上的免费在线教程。


将z-stack图像上传到Imaris 8.0.2软件。
点击“编辑/添加频道”,然后选择适当的图像。
在主菜单中,选择“替代/添加新曲面”,然后点击“ 创建曲面” 。
选择适当的“曲面区域细节级别”。选择“绝对强度”作为阈值。
注意:这两个设置都是自动计算的,但是,用户可以更改这些参数。在整个图像分析中,请保持这些设置不变。


单击“完成”以完成堆栈的3D重建。
通过添加新的斑点对象,创建3D斑点对象以量化核数。
选择“不同的光斑尺寸” 。
对于“源通道”,选择DAPI 通道。
对于“斑点检测”,输入原子核的“估计的XY直径和Z直径”。
手动调整“斑点质量”以包括任何未检测到的核。
选择“强度最大值=细胞轮廓的遮罩通道数”以限制额外的核斑点。请注意,所有描述的参数的值取决于所获取图像的属性,并且在特定图像中会有所不同。
调整“斑点区域阈值”,并确保检测到所有核斑点。
单击“完成”以完成对核斑点的检测。
选择所需的统计参数(斑点的数量和总数)并导出.csv文件。这些可以使用图形程序打开。
 


数据与分析
使用一个图形程序(Excel或的GraphPad)来计算每个表面(的体积总和微米3 )。
将数据表示为每个细胞的总表面体积(核斑)。
 


记事簿


 


细胞系选择:我们使用的LLC-PK 1 细胞系起源于欧洲已认证细胞培养物保藏中心。这LLC-PK 1 克隆本来也可以在ATCC(ATCC ® CRL-1392 TM ),但是已经停产。ATCC分布另一种LLC-PK 1 细胞系,但是,我们发现来自各种来源的LLC-PK 1 细胞的claudin-2表达水平有所不同。因此,在为该项目选择细胞系之前,应验证目标claudin的良好表达。
细胞计数和融合:随着层的融合,TJ 逐渐发展,因此建议使用完全融合的层。Claudin-2的总表达在亚汇合细胞中低,并随着汇合的发展而增加。比较不同条件时,汇合度应保持恒定。我们发现,当在培养和接种过程中持续应用相同的条件时,细胞数可以保持恒定。因此,在建立协议之后,不需要常规的细胞计数。但是,实验操作可能会改变细胞生长,因此应常规检查融合度,并将其视为重要变量。
在整个染色过程中,必须避免干燥盖玻片。
这里描述的协议用于染色claudin-2。可以使用相同的协议来可视化任何上皮细胞系中的其他连接蛋白。我们发现固定模式是良好保存和可视化连接蛋白的关键。当对claudins染色时,使用甲醇获得了良好的结果。对于occludin,我们使用丙酮固定。对于这些固定方法,不需要其他通透性。有关用于连接蛋白的各种固定方法的详细分析,请参阅Buckley 等。,2018 。
所描述的量化不是针对TJ定位的claudin-2,而是反映了总claudin-2的丰度,包括胞质蛋白。在我们的实验中,定量用于对照和TNF-α处理的样品之间的比较。相对于对照(未处理),数据表示为染色/细胞,以表明倍数变化。在这种情况下,对照未经处理的样品用作参考。对于其它类型的比较(例如,未经处理的样品的比较),参考蛋白质,例如,另一个TJ蛋白应使用(例如,costaining 用于闭合蛋白或ZO-1和表达紧密连接蛋白2的比率,以闭合蛋白染色)。基准的选择然而可以构成为细胞因子处理的样品的困难,因为我们发现,我们测试的所有TJ蛋白的表达(例如,其他密蛋白,闭合蛋白),以及所述粘着连接蛋白E-钙粘均改变由TNFα (Amoozadeh 等人,2015)。因此,在选择参考之前,重要的是要表明它不受治疗的影响。最后,应通过其他定量方法(例如s Western印迹)验证定量。
 


菜谱


 


细胞培养基
低糖,含L-谷氨酰胺和110 mg / L 丙酮酸钠的Dulbecco改良Eagle Eagle培养基(D-MEM)补充有10%胎牛血清(FBS)和无菌过滤的1%青霉素-链霉素


1x PBS
从10x PBS稀释:将50 ml 10x PBS加入450 ml蒸馏水2 O中


阻塞缓冲区
3%BSA和PBS


混合1.5克BSA和50毫升PBS
短期存放(1天)于4°C
经度不推荐克长期储存


抗体缓冲液
0.3%BSA和PBS


混合0.15 g BSA和50 ml PBS
短期存放(1天)于4°C
经度不推荐克长期储存


 


致谢


 


资金来源:加拿大肾脏基金会,加拿大卫生研究院(CIHR)授予PJT-149058和MOP-142409。


该协议源自:Amoozadeh 等。,2015年和2018年;Dan 等。,2019 。


 


利益争夺


 


作者宣称他们没有财务或非财务利益冲突。


参考资料


 


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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Anwer, S. and Szászi, K. (2020). Immunofluorescent Staining of Claudin-2 in Cultured Kidney Tubular Cells. Bio-protocol 10(14): e3678. DOI: 10.21769/BioProtoc.3678.
  2. Dan, Q., Shi, Y., Rabani, R., Venugopal, S., Xiao, J., Anwer, S., Ding, M., Speight, P., Pan, W., Alexander, R. T., Kapus, A. and Szászi, K. (2019). Claudin-2 suppresses GEF-H1, RHOA, and MRTF, thereby impacting proliferation and profibrotic phenotype of tubular cells. J Biol Chem 294(42): 15446-15465.
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