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Oct 2017
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Magnetic Resonance Imaging and Histopathological Visualization of Human Dural Lymphatic Vessels
人类硬脑膜淋巴管的磁共振成像及组织病理学观察   

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Abstract

In this protocol, we describe a method to visualize and map dural lymphatic vessels in-vivo using magnetic resonance imaging (MRI) and ex-vivo using histopathological techniques. While MRI protocols for routine imaging of meningeal lymphatics include contrast-enhanced T2-FLAIR and T1-weighted black-blood imaging, a more specific 3D mapping of the lymphatic system can be obtained by administering two distinct gadolinium-based MRI contrast agents on different days (gadofosveset and gadobutrol) and subsequently processing images acquired before and after administration of each type of contrast. In addition, we introduce methods for optimal immunostaining of lymphatic and blood vessel markers in human dura mater ex-vivo.

Keywords: Lymphatic vessels (淋巴管), Brain (脑), Meninges (脑膜), MRI (MRI), Histopathology (组织病理学), Immunohistochemistry (免疫组化)

Background

Among the causes of immune privilege in the brain is the absence of parenchymal lymphatic vessels. However, recent studies have uncovered an extensive lymphatic circulating system in the dura mater of rodents (Aspelund et al., 2015; Louveau et al., 2015), providing possible routes for the elimination of the brain’s waste products and for immune cells to access the deep cervical lymph nodes. In this protocol, we describe a way to: (1) visualize the lymphatic vessels in-vivo in the dura mater using MRI of the head, and (2) assess the local presence of lymphatic vessels using optimized immunostaining methods (Absinta et al., 2017). In-vivo imaging of lymphatics may enable more detailed studies of mechanisms of waste removal and immune function and their potential abnormalities in various diseases and aging.

Materials and Reagents

  1. Superfrost Plus Microslides (Daigger Scientific, catalog number: EF15978Z )
  2. Cover Glasses (Daigger Scientific, catalog number: EF15972L )
  3. Paper towel (KCWW, Kimberly-Clack, catalog number: 05511 )
  4. Polypropylene Coplin jar (IHC World, catalog number: IW-2501 )
  5. Super HT PAP pen (Biotium, catalog number: 22006 )
  6. Gadavist, gadobutrol (0.1 mmol/kg body weight, i.v., Bayer Health Care, NDC 50419-325-12)
  7. Ablavar, gadofosveset (0.03 mmol/kg body weight, i.v., Lantheus Medical Imaging, NDC 11994-012-02)
  8. 10% Neutral Buffered Formalin Fixatives, methanol < 2% (Leica Biosystems, catalog number: 3800602 )
  9. Ethanol (Pharmaco-AAPER, catalog number: 111000200 )
  10. Target Retrieval Solution, pH 9 (Agilent Technologies, Dako, catalog number: S2367 )
  11. Target Retrieval Solution (Agilent Technologies, Dako, catalog number: S1699 )
  12. Tris buffered saline 10x, pH 7.4 (KD Medical, catalog number: RGF-3385 )
  13. Hydrogen Peroxide, 30% (Fisher Scientific, catalog number: H325-500 )
  14. Protein Block, Serum-Free (Agilent Technologies, Dako, catalog number: X0909 )
  15. LYVE1 antibody (Abcam, catalog number: ab36993 )
  16. Podoplanin (D2-40) antibody (Bio-Rad Laboratories, catalog number: MCA2543 )
  17. CD31 antibody (Abcam, catalog number: ab28364 )
  18. PROX1 antibody (AngioBio, catalog number: 11-002P )
  19. COUP-TF II antibody (R&D Systems, catalog number: PP-H7147-00 )
  20. CCL21 antibody (Abcam, catalog number: ab9851 )
  21. CD68 (KP-1) antibody (Thermo Fisher Scientific, Invitrogen, catalog number: MA5-13324 )
  22. CD3 antibody (Agilent Technologies, Dako, catalog number: A0452 )
  23. Antibody Diluent (Agilent Technologies, Dako, catalog number: S0809 )
  24. PV Poly-HRP Anti-Mouse IgG (Leica Biosystems, catalog number: PV6114 )
  25. PV Poly-HRP Anti-Rabbit IgG (Leica Biosystems, catalog number: PV6119 )
  26. ImmPRESSTM-AP Anti-Rabbit IgG (Vector Laboratories, catalog number: MP-5401 )
  27. ImmPRESSTM-AP Anti-Mouse IgG (Vector Laboratories, catalog number: MP-5402 )
  28. Goat anti-Mouse IgG, Alexa Fluor 488 (Thermo Fisher Scientific, Invitrogen, catalog number: A-11029 )
  29. Goat anti-Rabbit IgG, Alexa Fluor 594 (Thermo Fisher Scientific, Invitrogen, catalog number: A-11012 )
  30. DAB substrate kit (Abcam, catalog number: ab94665 )
  31. Vector Blue Alkaline Phosphatase Substrate Kit (Vector Laboratories, catalog number: SK-5300 )
  32. Hematoxylin 560 MX (Leica Biosystems, catalog number: 3801575 )
  33. Blue buffer 8 (Leica Biosystems, catalog number: 3802916 )
  34. VectaMount Permanent Mounting Medium (Vector Laboratories, catalog number: H-5000 )
  35. Fluoro-Gel II Mounting Medium (Electron Microscopy Sciences, catalog number: 17985-50 )
  36. Tween 20 (Agilent Technologies, Dako, catalog number: S1966 )
  37. TBS-0.5% Tween 20 (TBST) (see Recipes)

Equipment

  1. 18-22 gauge catheter (Smiths Medical)
  2. Pressure infusion tubing (ICU Medical)
  3. Automatic pressure injector (Bayer, model: Medrad® Spectris Solaris® EP MR Injection System )
  4. 3-tesla MRI scanner unit (Siemens Skyra, Siemens Healthcare)
  5. 32-channel head coil for MRI signal reception (Siemens Skyra, Siemens Healthcare)
  6. Water bath (Leica Biosystem, model: Leica HI1210 )
  7. Humidified chamber (Simport, model: StainTrayTM M920 )
  8. Manual Rotary Microtome (Leica Biosystem, model: Leica RM2235 )
  9. Leica RM CoolClampTM (Leica Biosystem, model: Leica RM CoolClamp )
  10. Steamer (IHC World, model: IHC-TekTM Epitope Retrieval Steamer Set )
  11. Digital rocker (VWR, catalog number: 12620-906 )
  12. Microscope (Carl Zeiss, model: AxioObserver Z.1 )
  13. Microscope camera (Carl Zeiss, model: Axiocam 503 )
  14. Magnetic stirrer

Software

  1. MIPAV software (https://mipav.cit.nih.gov/)
  2. OsiriX software (http://www.osirix-viewer.com/)
  3. Zeiss Zen 2 Blue edition (https://www.zeiss.com/microscopy/int/products/microscope-software/zen.html)

Procedure

  1. Ethical approval
    All human research was carried out under an Institutional Review Board approved protocol, after obtaining informed consent. Formalin-fixed human dura was retrieved at autopsy after obtaining appropriate consent.

  2. Human imaging
    1. Place an intravenous line using an 18-22 gauge catheter and pressure infusion tubing linked to an MRI compatible automatic pressure injector. (Figure 1) 


      Figure 1. MRI preparation. Auto injector setup showing the injector (A), linked to the catheter through extension tubing (B, C), and the setup before the subject is moved into the MRI for scanning (D).

    2. Set up the subject in the MRI scanner with a 32-channel head coil.
    3. Perform cranial MRI as following sequences, a quoted method from Absinta et al. (2017).
      1. Whole-brain T1-Magnetization Prepared Rapid Acquisition of Gradient Echoes (MPRAGE, sagittal 3D turbo-fast low angle shot sequence, acquisition matrix 256 x 256, isotropic resolution 1 mm, 176 slices, repetition time [TR]/echo time [TE]/inversion time [TI] = 3,000/3/900 msec, flip angle 9°, acquisition time 5 min 38 sec).
      2. Limited T2-weighted Fluid Attenuation Inversion Recovery (FLAIR, coronal 2D acquisition over the superior sagittal sinus, field-of-view 256 mm2, 22 slices, reconstructed in-plane resolution 0.25 mm2, 42 contiguous 3 mm slices, TR/TE/TI = 6,500/93/2,100 msec, echo train length 17, bandwidth 80 Hz/pixel, acquisition time 5 min), optimized for detection of gadolinium-based contrast agent in the subarachnoid space.
      3. Black-blood scan (coronal acquisition, Sampling Perfection with Application optimized Contrasts using different flip angle Evolution [SPACE] sequence, field-of-view 174 mm2, matrix 320 x 320, reconstructed in-plane resolution 0.27 mm2, 64 contiguous 0.5 mm sections, TR/TE = 938/22 msec, echo train length 35, bandwidth 434 Hz/pixel, acquisition time 7 min 50 sec). Acquire a series of 2 or three overlapping coronal acquisitions to cover most of the cerebral hemispheres.
      4. Whole-brain T2-FLAIR scan (coronal 3D SPACE sequence, field-of-view 235 mm2, matrix 512 x 512, reconstructed in-plane resolution 0.46 mm2, 176 1 mm sections, TR/TE/TI = 4,800/354/1,800 msec, nonselective inversion pulse, echo-train length 298, bandwidth 780 Hz/pixel, acceleration factor 2, acquisition time 14 min).
      5. Whole-brain T1-SPACE (axial 3D acquisition, acquisition matrix 256 x 256, isotropic resolution 0.9 mm, 112 sections, TR/TE = 600/20 msec, flip angle 120°, echo-train length 28, acquisition time 10 min).
    4. Inject MRI contrast agent, either gadobutrol (0.1 mmol/kg body weight, i.v., Bayer HealthCare) or another standard agent, at a rate of 0.3 ml/min followed by 10 ml of saline flush.
    5. Repeat MRI sequences A3a, A3c, and A3d after completion of the infusion.
    6. Covert scanner-generated DICOM images into NIFTI files for processing using dcm2nii script (nitrc.org, open source).
    7. Co-register pre- and post-contrast images, perform skull-stripping, and subtract pre-contrast images from post-contrast images using standard algorithms implemented in MIPAV software (select Algorithms/Registration/Optimized Automatic Registration and Utilities/Image Calculator/Subtract, respectively).
    8. Import subtraction images into OsiriX software for maximum intensity projection (MIP) 3D rendering (select 2D/3D and then 3D Surface Rendering). (Figure 2)


      Figure 2. MRI visualization of dural lymphatic vessels in human. On post-gadobutrol coronal T2-FLAIR, the dura does not enhance, and lymphatic vessels (red arrows), running alongside the venous dural sinuses and within the falx cerebri, can be appreciated. 3D rendering, using OsiriX software, of putative dural lymphatics (black) in a 47-year old woman, derived from whole-brain T1-weighted SPACE MRI. (Modified from Figure 1 and Figure S1 in Absinta et al. [2017]. Creative Commons Attribution License)

    9. For more specific lymphatic imaging, perform Steps B1-B8 using gadofosveset (0.03 mmol/kg body weight, i.v., Lantheus Medical Imaging) rather than gadobutrol. Compare the subtraction images obtained from gadofovest and gadobutrol experiments to identify the lymphatic vessels (Figure 3).


      Figure 3. Gadobutrol vs. gadofosveset in MRI visualization of dural lymphatic vessels. Coronal T1-weighted black-blood images were acquired after intravenous injection of two different gadolinium-based contrast agents during two MRI sessions separated by one week. Dural lymphatics (red arrows in magnified view boxes) were better discerned using gadobutrol (standard MRI contrast agent, which readily enters the dura) compared to gadofosveset (serum albumin-binding contrast agent, which remains largely intravascular) and were localized around dural sinuses, middle meningeal artery, and cribriform plate (white arrows). Notably, the choroid plexus (white arrows) enhanced less with gadofosveset than gadobutrol, whereas meningeal and parenchymal blood vessels (both veins and arteries) did not enhance with any contrast agent and appeared black. (Originally published in Absinta et al. (2017). Creative Commons Attribution License)

  3. Immunohistochemistry, single staining
    1. Fix freshly dissected human dura mater with 10% formalin for 24-48 h at room temperature. Commercial 10% neutral buffered formalin (NBF) contains a small percentage of methanol as a stabilizer, which is not a problem for the majority of procedures. Dura should be fixed as soon as possible using gentle agitation (swirling) of the specimen to aid penetration and fixation reaction. Tissue should be fixed for 24-48 h in NBF, and then stored in 1x PBS with a few drops of 10% formalin at room temperature.
    2. Trim the dura into coronal sections and embed the tissue in a paraffin block (see Figure 4). Our recommendation is to focus on the coronal sections near the superior sagittal sinus, which can be easily identified in the dura.


      Figure 4. Whole-mount and coronal sections of the human dura mater for histological analysis. A. The red dotted line shows the sampling direction. B, C, and D. Show the coronal view of the dura mater sample before tissue processing.

    3. Using rotary microtome, cut the paraffin-embedded tissue block into sections of 3-8 µm thickness. Float the sections in 20% ethanol at room temperature, then transfer them to a 44 °C water bath. (see Note 1 and Video 1)

      Video 1. Demonstration of the sectioning of the human dura mater using a microtome. Before sectioning, place the paraffin tissue block surface on melting ice or cold wet paper towel. After sectioning, place the section in 20% ethanol and then into a warm floating bath.

    4. Transfer the sections onto Superfrost Plus Microslides, as uncoated or uncharged slides may not retain the tissue. Before drying out the slides, remove residual water using a snap of the wrist (imagine wielding a whip), which is important to prevent sections from lifting from slides. Allow the slides to dry vertically overnight, at room temperature, to allow trapped water to escape downward.
    5. Deparaffinize slides using xylene (3 changes of xylene, each 3 min).
    6. Rehydrate slides using 100% alcohol (3 changes, each 3 min), 80% alcohol (3 min) and 50% alcohols (3 min), respectively.
    7. Rinse slides in deionized water for 1 min.
    8. Perform heat-induced antigen retrieval to unmask the antigenic epitope using a steamer. Add tap water to the water base, to the “Max” line, and put the steaming plate onto the water reservoir. Fill a plastic Coplin jar with Target Retrieval Solution or Target Retrieval Solution, at pH 9, and dip deparaffinized/rehydrated slides in the jar. Place the plastic Coplin jar in the steamer and cover it. Turn on the steamer and set the timer for 20 min to incubate it at 95-100 °C. We recommend steamer for heat-induced antigen retrieval instead of microwave or pressure cooker, because it reduces the chance of the section falling off the slide.
    9. Take out the Coplin jar and allow it to cool down for 10 min at room temperature.
    10. Rinse slides gently in Tris-buffed saline (TBS) for 5 min. Use TBS or TBS-0.5% Tween 20 (TBST) during slide washing to prevent sections from falling off.
    11. Immerse sections in 0.3% H2O2 solution in deionized water at room temperature for 10 min to block endogenous peroxidase activity.
    12. Rinse slides gently in TBST for 1 min.
    13. Draw the hydrophobic barrier around the tissues using PAP pen.
    14. Rinse slides gently in TBST 20 for 1 min.
    15. Drop 3-4 of Dako Protein Block on the tissue and incubate at room temperature for 20 min in a humidified chamber.
    16. Gently drop off the excess Dako Protein Block from the slides. Do not rinse the slides in this step.
    17. Apply primary antibody + Dako Antibody Diluent (see Table 1 for antibody dilution factor; 100-200 µl is required to cover the tissue) on the tissues, and incubate at room temperature for 2 h or at 4 °C overnight in a humidified chamber. Make sure that the antibody is spread well on the tissues.

      Table 1. Condition of antigen retrieval, antibody dilution and time of incubation


    18. Wash slides in TBST 3 times, 5 min each, using a rocker.
    19. Apply secondary antibody (HRP anti-Mouse IgG or HRP anti-rabbit IgG) on the tissues and incubate for 30 min at room temperature in a humidified chamber.
    20. Wash slides in TBST 3 times, 5 min each, using a rocker.
    21. Drip 3-4 drops of freshly made DAB substrate solution on the slide and check the brown color of antibody signal by microscopy.
    22. If the staining reveals adequate intensity, stop the DAB reaction by dipping slides in deionized water. Over-staining will lead to high background that will obscure the true signals.
    23. Dip slides in Leica Hematoxylin 560 MX for 10 sec, for better morphology and contrast.
    24. Rinse slides in tap water for 5 min.
    25. Immerse slides in bluing solution (Leica Blue buffer or 0.2% ammonia solution or 0.1% lithium carbonate solution).
    26. Dehydrate slides through air dry and coverslip using Permount mounting solution. The mounted slides can be kept at room temperature constantly. 

  4. Immunohistochemistry, double staining of D2-40 and CD31 (simultaneous double staining of lymphatic and blood vessels, respectively)
    1. Follow Steps C5-C16 above.
    2. Apply cocktails of primary antibodies + Dako Antibody Diluent on the tissues and incubate at room temperature for 2 h in a humidified chamber.
    3. Wash slides in TBST 3 times, 5 min each, using a rocker.
    4. Apply secondary antibody (HRP anti-Mouse IgG for D2-40 and ImmPRESSTM-AP anti-Rabbit IgG for CD31) on the tissues and incubate for 30 min at room temperature in a humidified chamber.
    5. Wash slides in TBST 3 times, 5 min each, using a rocker.
    6. Drip 3-4 drops of freshly made DAB substrate solution on the slide and check the brown color of D2-40 antibody signal by microscopy.
    7. Wash slides in deionized water to stop the DAB reaction.
    8. Drip 3-4 drops of fresh Vector Blue substrate solution on the same slide and check the blue color of CD-31 antibody signal by microscopy.
    9. If the staining reveals enough intensity, stop the Vector Blue reaction by dipping slides in deionized water.
      CAUTION: Do NOT perform hematoxylin counterstaining following use of the Vector Blue chromogen.
    10. Dehydrate slides through air dry and coverslip using Permount mounting solution.

  5. Immunohistochemistry, double staining of PROX1 and CD31 (sequential double staining)
    1. Follow Steps C5-C22 above. Finish PROX1 immunostaining without counterstaining.
    2. Drip 3-4 drops of Dako Protein Block on the tissue and incubate at room temperature for 20 min in a humidified chamber.
    3. Gently drop off the excess Dako Protein Block from the slides. Do not rinse the slides in this step.
    4. Apply CD31 antibodies + Dako Antibody Diluent on the tissues and incubate at room temperature for 2 h in a humidified chamber.
    5. Wash slides in TBST 3 times, 5 min each, using a rocker.
    6. Apply secondary antibody (ImmPRESSTM-AP anti-Rabbit IgG for CD31) on the tissues and incubate for 30 min at room temperature in a humidified chamber.
    7. Wash slides in TBST 3 times, 5 min each, using a rocker.
    8. Drip 3-4 drops of fresh Vector Blue substrate solution on the same slides and check the blue color of CD-31 antibody signal by microscopy.
    9. Stop the Vector Blue reaction by dipping slides in deionized water if the staining reveals enough intensity.
      CAUTION: Do NOT perform hematoxylin counterstaining following use of the Vector Blue chromogen.
    10. Dehydrate slides through air drying and coverslip using Permount mounting solution.

  6. Immunofluorescence, double staining of D2-40 + CD31 (simultaneous double staining)
    1. Follow Steps D1-D3 above.
    2. Apply cocktails of secondary antibodies (Goat anti-Mouse IgG Alexa Fluor 488 and Goat anti-rabbit IgG Fluor 594, 1:200 diluted in Dako Antibody Diluent) on the tissues and incubate for 30 min at room temperature in a humidified chamber.
    3. Wash slides in TBST 3 times, 5 min each, using a rocker.
    4. Dehydrate slides through air dry and coverslip using Fluoro-Gel II Mounting Medium.
    5. Observe the localization of D2-40 and CD31 with fluorescence microscopy.

Data analysis

Scan the entire slide and stitch it together by greater than 10x magnification using Zeiss Microscope, camera, and Zeiss Zen Blue software. On slides double-stained for lymphatic and vascular endothelial markers (D2-40/CD31 and PROX1/CD31), identify lymphatic structures and mark them on the screen under the microscope using the following criteria: (a) structures of endothelial cell-lined vessel; (b) vessel with thin endothelial cells, the nuclei of cell bulge into the lumen; (c) semi-collapsed thin vessel wall with poor basal lamina; and (d) no or only a few red blood cells in the lumen of the vessel (Killer et al., 2008). Lymphatic vessels are counted, and their dimensions are measured. If samples vary in disease type or treatment status, simple comparative statistics may be computed on the count and diameter data (Figure 5).


Figure 5. Neuropathology of human dural lymphatic vessels, coronal section. A, B and C. Within the dura mater, lymphatic and blood vessels can be differentiated using double staining for PROX1 (a transcription factor involved in lymphangiogenesis, nuclear staining) and CD31 (a vascular endothelial cell marker). E, F and G. Similarly, lymphatic and blood vessels can be differentiated using double staining for D2-40 (endothelial membrane staining) and CD31. Red blood cells are seen within blood vessels, but not within lymphatic vessels. D and H. Using Zeiss Zen Blue software, lymphatic structures are marked on the digitalized slide. Insets (B, C, F, G) were rotated relative to the original Figures in A and E. Scale bars: 1 mm (A, G), 100 μm (B, C, F, G). Abbreviations: LV–lymphatic vessels; BV–blood vessels. (Modified from Figure 3 in Absinta et al. [2017]. Creative Commons Attribution License)

Notes

  1. Human dura mater is a very tough tissue, and microtome sectioning is difficult. Chilling the paraffin blocks (e.g., Leica RM Cool ClampTM) makes sectioning of dura easier. Also, when tissue is exposed on the surface of a paraffin block by rough trimming, it has the capacity to absorb water, which can penetrate a small distance into the tissue, resulting in softening and swelling it. For the dura mater, this effect may allow a couple of sections to be cut easily. By placing the trimmed block surface on melting ice or in a tray of ice water at 4 °C for 1 min, followed by use of a cold wet paper towel for 30 sec to 1 min, the sectioning becomes easier. Generally, after this procedure, the best quality sections are achieved by cutting very slowly.
  2. Paraffin sections of dura may wrinkle easily, which can generate artifacts and ultimately nonspecific staining. Non-standard flotation techniques may be useful if the sections obtained from a block are highly wrinkled. If sections are initially floated in 20% ethanol then transferred, on a slide, to a hot flotation bath, the wrinkling may be mitigated. 20% ethanol actively removes the wrinkles out because it has lower surface tension than water.
  3. Formalin fixed-paraffin embedded (FFPE) human skin can be used as a positive control for lymphatic vessel marker and assessment. FFPE Hippocampus (CA3) of brain tissue can be used as good positive control for PROX1 staining.

Recipes

  1. TBS-0.5% Tween 20 (TBST)
    200 ml 10x TBS
    1,800 ml deionized water
    Add 1 ml of Tween 20, mixed well using a magnetic stirrer

Acknowledgments

The Intramural Research Program of NINDS supported this study. This protocol was adapted from procedures published in Absinta et al. (2017). Figures 2, 3, and 5 were modified and reproduced with permission from Absinta et al. (2017). The authors declare no conflicts of interest.

References

  1. Absinta, M., Ha, S. K., Nair, G., Sati, P., Luciano, N. J., Palisoc, M., Louveau, A., Zaghloul, K. A., Pittaluga, S., Kipnis, J. and Reich, D. S. (2017). Human and nonhuman primate meninges harbor lymphatic vessels that can be visualized noninvasively by MRI. eLife: e29738.
  2. Aspelund, A., Antila, S., Proulx, S. T., Karlsen, T. V., Karaman, S., Detmar, M., Wiig, H. and Alitalo, K. (2015). A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. J Exp Med 212(7): 991-999.
  3. Louveau, A., Smirnov, I., Keyes, T. J., Eccles, J. D., Rouhani, S. J., Peske, J. D., Derecki, N. C., Castle, D., Mandell, J. W., Lee, K. S., Harris, T. H. and Kipnis, J. (2015). Structural and functional features of central nervous system lymphatic vessels. Nature 523(7560): 337-341.
  4. Killer, H. E., Jaggi, G. P., Miller, N. R., Flammer, J. and Meyer, P. (2008). Does immunohistochemistry allow easy detection of lymphatics in the optic nerve sheath? J Histochem Cytochem 56(12): 1087-92.

简介

在该协议中,我们描述了一种使用组织病理学技术使用磁共振成像(MRI)和体外可视化和绘制硬膜淋巴管体内的方法。 虽然用于脑膜淋巴管的常规成像的MRI方案包括对比增强的T2-FLAIR和T1加权的黑血成像,但是可以通过在不同日期施用两种不同的基于钆的MRI造影剂来获得更具体的淋巴系统3D图谱 (gadofosveset和钆布醇)并且随后处理在施用每种类型的对比之前和之后采集的图像。 此外,我们介绍了在人体硬脑膜内进行淋巴和血管标志物最佳免疫染色的方法。

【背景】在大脑免疫特权的原因是没有实质淋巴管。 然而,最近的研究发现了啮齿动物硬脑膜中广泛的淋巴循环系统(Aspelund等人,2015; Louveau等人,2015),提供了可能的途径 用于消除大脑的废物和免疫细胞进入深部颈部淋巴结。 在这个协议中,我们描述了一种方法来:(1)使用MRI的头部显示硬脑膜内的淋巴管体内,和(2)使用优化评估淋巴管的局部存在 免疫染色方法(Absinta等人,2017)。 淋巴管的体内成像可以更详细地研究废物清除和免疫功能的机制及其在各种疾病和衰老中的潜在异常。

关键字:淋巴管, 脑, 脑膜, MRI, 组织病理学, 免疫组化

材料和试剂

  1. Superfrost Plus Microslides(Daigger Scientific,目录号:EF15978Z)
  2. 封面眼镜(Daigger Scientific,目录号:EF15972L)
  3. 纸巾(KCWW,Kimberly-Clack,目录号:05511)
  4. 聚丙烯Coplin广口瓶(IHC World,目录号:IW-2501)
  5. 超高温PAP笔(Biotium,目录号:22006)
  6. Gadavist,钆布醇(0.1 mmol / kg体重,i.v.,Bayer Health Care,NDC 50419-325-12)
  7. Ablavar,gadofosveset(0.03 mmol / kg体重,i.v.,Lantheus Medical Imaging,NDC 11994-012-02)
  8. 10%中性缓冲福尔马林固定剂, 2%(Leica Biosystems,目录号:3800602)
  9. 乙醇(Pharmaco-AAPER,目录号:111000200)
  10. 目标检索解决方案,pH 9(安捷伦科技公司,Dako,目录号:S2367)
  11. 目标检索解决方案(Agilent Technologies,Dako,目录号:S1699)
  12. Tris缓冲盐水10倍,pH 7.4(KD Medical,目录号:RGF-3385)
  13. 过氧化氢,30%(Fisher Scientific,目录号:H325-500)
  14. 蛋白质块,无血清(安捷伦科技公司,Dako,产品目录号:X0909)
  15. LYVE1抗体(Abcam,目录号:ab36993)
  16. Podoplanin(D2-40)抗体(Bio-Rad Laboratories,目录号:MCA2543)
  17. CD31抗体(Abcam,目录号:ab28364)
  18. PROX1抗体(AngioBio,目录号:11-002P)
  19. COUP-TF II抗体(R&amp; D Systems,目录号:PP-H7147-00)
  20. CCL21抗体(Abcam,目录号:ab9851)
  21. CD68(KP-1)抗体(Thermo Fisher Scientific,Invitrogen,目录号:MA5-13324)
  22. CD3抗体(Agilent Technologies,Dako,目录号:A0452)
  23. 抗体稀释剂(Agilent Technologies,Dako,产品目录号:S0809)
  24. PV Poly-HRP抗小鼠IgG(Leica Biosystems,目录号:PV6114)
  25. PV Poly-HRP抗兔IgG(Leica Biosystems,目录号:PV6119)
  26. ImmPRESS TM -AP抗兔IgG(Vector Laboratories,目录号:MP-5401)
  27. ImmPRESS TM -AP抗 - 小鼠IgG(Vector Laboratories,目录号:MP-5402)
  28. 山羊抗小鼠IgG,Alexa Fluor 488(Thermo Fisher Scientific,Invitrogen,目录号:A-11029)
  29. 山羊抗兔IgG,Alexa Fluor 594(Thermo Fisher Scientific,Invitrogen,目录号:A-11012)
  30. DAB底物试剂盒(Abcam,目录号:ab94665)
  31. Vector Blue碱性磷酸酶底物试剂盒(Vector Laboratories,产品目录号:SK-5300)
  32. 苏木素560 MX(Leica Biosystems,产品目录号:3801575)
  33. 蓝色缓冲液8(Leica Biosystems,目录号:3802916)
  34. VectaMount永久性安装介质(Vector Laboratories,目录号:H-5000)
  35. Fluoro-Gel II安装介质(Electron Microscopy Sciences,目录号:17985-50)
  36. 吐温20(安捷伦科技公司,Dako,目录号:S1966)
  37. TBS-0.5%吐温20(TBST)(见食谱)

设备

  1. 18-22号导管(史密斯医疗)
  2. 压力输液管(ICU医疗)
  3. 自动压力注射器(Bayer,型号:Medrad®)Spectris Solaris EP MR注射系统)
  4. 3-tesla MRI扫描仪单元(西门子Skyra,西门子医疗)
  5. 用于MRI信号接收的32通道头线圈(西门子Skyra,西门子医疗)
  6. 水浴(徕卡生物系统,型号:徕卡HI1210)
  7. 加湿室(Simport,型号:StainTray TM M920)
  8. 手动旋转切片机(徕卡生物系统,型号:Leica RM2235)
  9. Leica RM CoolClamp TM(Leica Biosystem,型号:Leica RM CoolClamp)
  10. 蒸锅(IHC World,型号:IHC-Tek TM表位检索蒸锅组)。
  11. 数字摇杆(VWR,目录号:12620-906)
  12. 显微镜(卡尔蔡司,型号:AxioObserver Z.1)
  13. 显微镜照相机(卡尔蔡司,型号:Axiocam 503)
  14. 磁力搅拌器

软件

  1. MIPAV软件( https://mipav.cit.nih.gov/ )
  2. OsiriX软件( http://www.osirix-viewer.com/ )
  3. Zeiss Zen 2蓝色版( https://www.zeiss.com /microscopy/int/products/microscope-software/zen.html )

程序

  1. 道德批准
    所有人类研究都是在获得知情同意后,根据机构审查委员会批准的方案进行的。
    在获得适当的同意后,在尸体解剖中检索福尔马林固定的人类硬脑膜
  2. 人体成像
    1. 使用18-22规格的导管和压力灌注导管放置静脉导管,导管与MRI兼容的自动压力注射器连接。 (图1)


      图1. MRI准备。自动注射器设置显示注射器(A),通过延伸管(B,C)连接到导管,以及在对象移入MRI进行扫描之前的设置D)。


    2. 使用32通道头部线圈在MRI扫描仪中设置对象
    3. 按照以下顺序执行头颅MRI,这是Absinta等人引用的方法。(2017)。
      1. 全脑T1磁化准备快速获取梯度回波(MPRAGE,矢状三维涡轮快速低角度拍摄序列,采集矩阵256 x 256,各向同性分辨率1 mm,176个切片,重复时间[TR] /回波时间[TE] /倒置时间[TI] = 3,000 / 3/900毫秒,翻转角9°,采集时间5分38秒);
      2. 有限的T2加权液体衰减反转恢复(FLAIR,在上矢状窦上的冠状面二维采集,视场256mm2,22个切片,重构的平面内分辨率0.25mm2 42个连续的3mm切片,TR / TE / TI = 6,500 / 93 / 2,100毫秒,回波序列长度17,带宽80Hz /像素,采集时间5分钟),优化用于检测钆基造影剂在蛛网膜下腔;
      3. 黑色血液扫描(采用不同的翻转角演变[SPACE]序列,视场174 mm 2,矩阵320 x 320,重建的平面分辨率0.27mm 2,64个连续的0.5mm部分,TR / TE = 938/22毫秒,回波串长度35,带宽434Hz /像素,采集时间7分50秒)。
        获取一系列2或3个重叠的冠状位采集,覆盖大部分大脑半球。
      4. 全脑T2-FLAIR扫描(冠状三维空间序列,视场235mm2,矩阵512x512,重建的平面分辨率0.46mm2, 176 1毫米截面,TR / TE / TI = 4,800 / 354 / 1,800毫秒,非选择性反转脉冲,回波列长度298,带宽780赫兹/像素,加速因子2,采集时间14分钟);
      5. 全脑T1-SPACE(轴向3D采集,采集矩阵256×256,各向同性分辨率0.9mm,112段,TR / TE = 600/20毫秒,翻转角120°,回波序列长度28,采集时间10分钟) 。
    4. 注射MRI造影剂,钆布醇(0.1 mmol / kg体重,静脉注射,拜耳医药保健)或其他标准剂,速度为0.3 ml / min,然后注射10 ml生理盐水。

    5. 完成输液后重复MRI序列A3a,A3c和A3d。
    6. 使用dcm2nii脚本( nitrc.org ,开源)将扫描器生成的DICOM图像隐藏到NIFTI文件中进行处理。
    7. 使用在MIPAV软件中实现的标准算法(选择算法/注册/优化的自动注册和实用程序/图像计算器/减法),共注册前和后对比图像,执行头颅剥离,并从后对比图像中减去预对比图像, 分别)。
    8. 将相减图像导入OsiriX软件以进行最大强度投影(MIP)3D渲染(选择2D / 3D,然后选择3D表面渲染)。 (图2)


      图2.人类硬脑膜淋巴管的MRI可视化在钆布醇后的冠状T2-FLAIR上,硬膜并不增强,淋巴管(红色箭头)与静脉硬脑膜窦一起运行,并在其内部镰刀大脑,可以赞赏。使用OsiriX软件对来自全脑T1加权SPACE MRI的47岁女性的推定硬脑膜淋巴管(黑色)进行3D渲染。

    9. 对于更具体的淋巴成像,使用gadofosveset(0.03 mmol / kg体重,i.v.,Lantheus Medical Imaging)而不是钆布醇执行步骤B1-B8。比较从gadofovest和钆布醇实验获得的减影图像,以确定淋巴管(图3)。


      图3.Gadobutrol与gadofosveset在硬脑膜淋巴管MRI可视化中的比较在两次MRI会话期间静脉注射两种不同的基于钆的造影剂后获得冠状T1加权黑色血图像,周。与gadofosveset(血清白蛋白结合造影剂,其主要保留血管内)相比,使用钆布醇(标准MRI造影剂,其容易进入硬脑膜)更好地辨别硬脑膜淋巴管(放大视图框中的红色箭头)并且位于硬膜窦周围,中脑膜动脉和筛板(白色箭头)。值得注意的是,脉络丛(白色箭头)用gadofosveset比钆布醇增加更少,而脑膜和实质血管(静脉和动脉)没有用任何造影剂增强并呈现黑色。

  3. 免疫组化,单染色
    1. 在室温下用10%福尔马林溶液新鲜解剖人类硬脑膜24-48小时。商用10%中性福尔马林缓冲液(NBF)含有少量甲醇作为稳定剂,这对于大多数程序来说不是问题。应尽可能快地固定Dura,并轻轻搅动(旋转)样本以帮助穿透和固定反应。组织应在NBF中固定24-48小时,然后在室温下储存在含有几滴10%福尔马林的1x PBS中。
    2. 将硬脑膜修剪成冠状切片并将组织嵌入石蜡块(见图4)。我们的建议是关注靠近上矢状窦的冠状面,这可以在硬脑膜中很容易识别。


      图4.人体硬脑膜的整体和冠状切片用于组织学分析。A.红色虚线显示取样方向。 B,C和D.在组织处理之前显示硬脑膜样本的冠状位图。

    3. 使用旋转切片机,将石蜡包埋的组织块切成3-8μm厚的切片。在室温下将部分在20%乙醇中漂浮,然后将它们转移到44℃水浴中。 (见注1和视频1)

      Video1
      视频1.使用切片机显示人体硬脑膜切片。在切片之前,将石蜡组织块表面放在融化的冰块或冷湿纸巾上。切片后,将切片置于20%乙醇中,然后放入温暖的漂浮浴中。

    4. 将部分转移到Superfrost Plus Microslides上,因为未涂层或不带电的载玻片可能无法保留组织。在干燥玻片前,用手指扣住残余的水分(想象挥舞着鞭子),这对防止玻片从玻片上抬起很重要。
      。让幻灯片在室温下垂直干燥过夜,让被困水流向下。

    5. 使用二甲苯去石蜡片(3次更换二甲苯,每次3分钟)。

    6. 使用100%酒精(3次更换,每3分钟),80%酒精(3分钟)和50%酒精(3分钟)分别补充幻灯片。
    7. 在去离子水中冲洗载玻片1分钟。
    8. 使用蒸汽锅进行热诱导的抗原修复以揭露抗原表位。将自来水加到水底,“Max”线上,并将蒸汽盘放到水箱上。用pH为9的目标回收溶液或目标回收溶液填充塑料Coplin瓶,并在罐中浸渍脱蜡/再水合的载玻片。将塑料Coplin瓶放入蒸锅并盖上。打开蒸锅并将计时器设定为20分钟,以在95-100℃孵育。我们推荐热敏抗原修复的蒸锅代替微波炉或压力锅,因为它可以减少切片从切片上脱落的机会。
    9. 取出Coplin罐,让其在室温下冷却10分钟。
    10. 用Tris缓冲盐水(TBS)轻轻冲洗5分钟。
      在洗涤过程中使用TBS或TBS-0.5%吐温20(TBST)以防止切片脱落。
    11. 将切片置于去离子水中的0.3%H 2 O 2溶液中室温10分钟以阻断内源性过氧化物酶活性。

    12. 在TBST中轻轻冲洗1分钟

    13. 使用PAP笔在组织周围绘制疏水屏障。
    14. 用TBST 20轻轻冲洗1分钟。
    15. 将3-4片Dako蛋白质块放在组织上,并在室温下在加湿室中孵育20分钟。
    16. 轻轻地将多余的Dako蛋白质块从载玻片上掉落下来。
      不要在这一步冲洗幻灯片。
    17. 在组织上应用第一抗体+ Dako抗体稀释剂(参见表1中的抗体稀释因子;需要100-200μl以覆盖组织),并在室温下孵育2小时或在4℃孵育过夜。确保抗体在组织上传播良好。

      表1.抗原提取条件,抗体稀释和孵育时间


    18. 使用摇杆在TBST中洗3次,每次5分钟。
    19. 在组织上涂上二抗(HRP抗小鼠IgG或HRP抗兔IgG),室温孵育30分钟。
    20. 使用摇杆在TBST中洗3次,每次5分钟。
    21. 在载玻片上滴3-4滴新鲜的DAB底物溶液,用显微镜检查抗体信号的棕色。
    22. 如果染色显示足够的强度,则通过将载玻片浸入去离子水中停止DAB反应。过度染色会导致高背景,从而掩盖真实的信号。
    23. 在Leica Hematoxylin 560 MX中浸10秒,以获得更好的形态和对比度。

    24. 冲洗自来水中的幻灯片5分钟
    25. 将蓝片溶液(徕卡蓝缓冲液或0.2%氨溶液或0.1%碳酸锂溶液)浸入玻片。
    26. 使用Permount安装解决方案通过空气干燥和盖玻片脱水滑道。安装的幻灯片可以保持室温不断。

  4. 免疫组织化学,D2-40和CD31的双染色(分别同时双重染色淋巴和血管)
    1. 按照上面的步骤C5-C16。
    2. 将一抗+ Dako抗体稀释剂的鸡尾酒应用于组织,并在室温下在潮湿的室内孵育2小时。
    3. 使用摇杆在TBST中洗3次,每次5分钟。
    4. 在组织上应用二抗(用于D2-40的HRP抗小鼠IgG和用于CD31的ImmPRESS TM -AP抗兔IgG),并在室温下在潮湿的室中温育30分钟。 />
    5. 使用摇杆在TBST中洗3次,每次5分钟。
    6. 在载玻片上滴3-4滴新制DAB底物溶液,用显微镜检查D2-40抗体信号的棕色。
    7. 在去离子水中清洗载玻片以停止DAB反应。
    8. 在相同的载玻片上滴3-4滴新鲜的Vector Blue底物溶液,用显微镜检查CD-31抗体信号的蓝色。
    9. 如果染色显示足够的强度,通过将载玻片浸入去离子水中停止Vector Blue反应。
      注意:使用Vector Blue色原体后,请勿执行苏木精复染。

    10. 使用Permount安装解决方案通过空气干燥和盖玻片脱水滑梯
  5. 免疫组织化学,PROX1和CD31的双重染色(连续双染色)
    1. 按照上面的步骤C5-C22。 PROX1免疫染色无复染。
    2. 滴3-4滴Dako蛋白质块在组织上,并在室温下孵育20分钟,在潮湿的房间里。
    3. 轻轻地将多余的Dako蛋白质块从载玻片上掉落下来。
      不要在这一步冲洗幻灯片。

    4. 在组织上涂抹CD31抗体+ Dako抗体稀释液并在室温下孵育2小时。
    5. 使用摇杆在TBST中洗3次,每次5分钟。

    6. 在室温下在湿润的室中孵育30分钟,在组织上应用二抗(用于CD31的ImmPRESS TM -AP抗兔IgG)。
    7. 使用摇杆在TBST中洗3次,每次5分钟。
    8. 在相同的载玻片上滴3-4滴新鲜Vector Blue底物溶液,用显微镜检查CD-31抗体信号的蓝色。

    9. 如果染色显示出足够的强度,请停止使用去离子水中的载玻片 注意:使用Vector Blue色原体后,请勿执行苏木精复染。
    10. 使用Permount安装解决方案通过空气干燥和盖玻片脱水滑道。

  6. 免疫荧光,D2-40 + CD31的双重染色(同时双重染色)
    1. 按照上述步骤D1-D3。
    2. 在组织上涂抹二抗(山羊抗小鼠IgG Alexa Fluor 488和山羊抗兔IgG Fluor 594,1:200在Dako Antibody Diluent中稀释)的鸡尾酒,并在室温下在湿润的培养箱中温育30分钟。 />
    3. 使用摇杆在TBST中洗3次,每次5分钟。

    4. 使用Fluoro-Gel II安装介质使空气干燥和盖玻片脱水
    5. 用荧光显微镜观察D2-40和CD31的定位。

数据分析

扫描整张幻灯片,使用Zeiss显微镜,相机和蔡司Zen Blue软件将其放大10倍以上。在淋巴和血管内皮标记物(D2-40 / CD31和PROX1 / CD31)上双重染色的载玻片上,鉴定淋巴结构,并使用以下标准将它们标记在显微镜下:(a)内皮细胞衬里血管; (b)具有薄内皮细胞的血管,细胞核膨胀进入内腔; (c)具有不良基底层的半塌陷薄壁血管;和(d)血管腔内没有或仅有少量红细胞(Killer等人,2008)。计数淋巴管,测量其尺寸。如果样本在疾病类型或治疗状态上有所不同,可以根据计数和直径数据计算简单的比较统计数据(图5)。


图5.人硬膜淋巴管的神经病理学,冠状切片A,B和C.在硬脑膜内,淋巴管和血管可以使用PROX1(参与淋巴管生成的转录因子)的双染色来区分,核染色)和CD31(血管内皮细胞标记物)。 E,F和G.类似地,使用D2-40(内皮细胞膜染色)和CD31的双染色可以区分淋巴和血管。红血细胞在血管内可见,但不在淋巴管内。 D和H.使用Zeiss Zen Blue软件,淋巴结构被标记在数字化幻灯片上。插图(B,C,F,G)相对于A和E中的原始图旋转。比例尺:1mm(A,G),100μm(B,C,F,G)。缩写:LV-淋巴管; BV-血管。

笔记

  1. 人类硬脑膜是一个非常艰难的组织,切片机切片困难。冷却石蜡块(例如,Leica RM Cool Clamp TM )可使硬脑膜切片更容易。此外,当通过粗糙修剪将组织暴露在石蜡块表面时,它具有吸收水的能力,水可以穿透组织进入一小段距离,导致其软化并膨胀。对于硬脑膜,这种效应可能允许几个部分容易切割。将切好的块表面置于融化的冰块或4℃的冰水盘中1分钟,然后使用冷湿纸巾30秒至1分钟,切片变得更容易。通常,在此过程之后,通过非常缓慢地切割来实现最佳质量部分。
  2. 硬脑膜石蜡切片可能容易起皱,这可能会产生伪像并最终导致非特异性染色。非标准浮选技术可能是有用的,如果从一个街区获得的部分高度起皱。如果部分最初漂浮在20%乙醇中,然后在载玻片上转移到热漂浮浴中,可以减轻起皱。 20%乙醇主动去除皱纹,因为它具有比水低的表面张力。
  3. 福尔马林固定石蜡包埋(FFPE)人皮肤可用作淋巴管标志物和评估的阳性对照。脑组织的FFPE海马(CA3)可用作PROX1染色的良好阳性对照。

食谱

  1. TBS-0.5%吐温20(TBST)
    200毫升10x TBS

    1800毫升去离子水 加入1毫升吐温20,使用磁力搅拌器充分混合

致谢

NINDS的校内研究计划支持这项研究。该协议是根据Absinta等人发表的程序改编的(2017年)。图2,图3和图5经过Absinta et al。(2017)的许可修改并转载。作者宣称没有利益冲突。

参考

  1. Absinta,M.,Ha,SK,Nair,G.,Sati,P.,Luciano,NJ,Palisoc,M.,Louveau,A.,Zaghloul,KA,Pittaluga,S.,Kipnis,J.and Reich,DS (2017年)。 人类和非人类灵长类动物脑膜具有淋巴管,MRI可非侵入性地显示淋巴管。 eLife :e29738。
  2. Aspelund,A.,Antila,S.,Proulx,S.T.,Karlsen,T.V.,Karaman,S.,Detmar,M.,Wiig,H.and Alitalo,K。(2015)。 引流脑间质液和大分子的硬脑膜淋巴管系统 J Exp Med 212(7):991-999。
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Copyright Ha et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ha, S., Nair, G., Absinta, M., Luciano, N. J. and Reich, D. S. (2018). Magnetic Resonance Imaging and Histopathological Visualization of Human Dural Lymphatic Vessels. Bio-protocol 8(8): e2819. DOI: 10.21769/BioProtoc.2819.
  2. Absinta, M., Ha, S. K., Nair, G., Sati, P., Luciano, N. J., Palisoc, M., Louveau, A., Zaghloul, K. A., Pittaluga, S., Kipnis, J. and Reich, D. S. (2017). Human and nonhuman primate meninges harbor lymphatic vessels that can be visualized noninvasively by MRI. eLife: e29738.
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lijun zhang
the First Affiliated Hospital, Sun Yat-sen University
Why is Target Retrieval Solution PH 9 when stained with LYVE-1 antibody. Looking forward to having your reply. Thanks very much.
2019/8/30 14:51:14 回复
Daniel Reich
Division of Neuroimmunology and Neurovirology, National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), USA, USA,

Thanks for your question about our protocol for immunohistochemistry (IHC) of formalin-fixed, paraffin-embedded (FFPE) tissue. Antigen retrieval is a critical point. Although formalin-based fixatives yield great preservation of morphology and tissue architecture, formalin fixation reduces the sensitivity of the IHC technique. Formaldehyde covalently binds to tissue protein and acts to crosslink adjoining proteins to form large accumulations of protein. The cross-linking of proteins to the antigen is supposed to “mask” the epitope and thus interfere with the binding of the antibody. So, antigen retrieval, such as heating (HIER) or enzyme processing, is necessary for FFPE tissue.

Although the mechanisms by which HIER acts are unknown, many pathologists have thought it turns back the formaldehyde-mediated chemical modifications of the antigen. Since the initial description of HIER, a wide range of buffered solutions (even “universal buffers”) have been developed. At present, HIER solutions can be divided two categories based on pH and buffer compositions, as low pH citrate buffer VS high pH EDTA buffer.

The classical theory is the knowledge that the heated efficiency of the citrate buffer-mediated HIER process breaks the cross-links that bind surrounding proteins to the antigen and open the epitope. On the other hand, EDTA buffer-mediated HIER is believed to work by removing bound calcium ions from the sites of cross-links. In this second theory, the chelation of calcium ions bound to proteins during fixation could be a critical step in HIER. Interestingly, EDTA buffers are particularly effective on over-fixed specimens and for the recovery of hard-to-detect antigens.

In our experience in long-fixed brain samples, high-pH EDTA-based solutions work well for some antigens (CD3, CD4, CD8, PD-1, PD-L1, LYVE-1, Prox1, D2-40) that are difficult to retrieve with citrate. Additionally, most phospho-tyrosine-specific antibodies appear to require the EDTA buffer (reference: Cell Signal Technology brochure). In our study of the dural lymphatic system, the EDTA buffer showed very specific signal with less background than citrate, so we recommend EDTA for LYVE-1 IHC in FFPE tissue.

2019/9/17 16:38:15 回复


Govind Nair
Govind Nair
There is an error in the injection rate mentioned in point #4 of Procedure section - the correct rate should be 0.3 ml/sec.
2019/6/21 6:50:20 回复