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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

Abstract

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in naïve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

Introduction

Recent advances in our understanding of cancer metastasis have uncovered that epithelial-to-mesenchymal transition (EMT) and the induction of a migratory/invasive cancer cell subpopulation are not, by themselves, sufficient for hematogenous dissemination1. Indeed, it was previously thought that metastasizing cancer cells intravasate through the entirety of cancer-associated endothelium as the tumor neovasculature is often characterized by low pericyte coverage, and as such, is highly permeable and unstable2,3,4. Although highly suggestive of defective functions within the tumor, vascular modifications during carcinogenesis do not provide evidence per se that tumor cells can penetrate blood vessels easily and in an uncontrolled fashion. Insights from intravital imaging (IVI) studies, in which tumor cells are fluorescently-tagged and the vasculature is labeled via the intravenous injection of fluorescent probes (such as dextran or quantum dots), show that, while tumor vessels are uniformly permeable to low molecular weight dextrans (e.g. 70 kD), high molecular weight dextrans (155 kD) and tumor cells can cross the endothelium only at specialized sites of intravasation which are preferentially located at vascular branch point5,6,7. Immunohistochemical (IHC) analyses using animal models and human patient-derived material have shown that these sites are "doorways" that specialize in regulating vascular permeability, locally and transiently, providing a brief window of opportunity for migratory/invasive cancer cells to enter the circulation. These doorways are called "Tumor Microenvironment of Metastasis" or "TMEM", and, quite expectedly, their density correlates with an increased risk of developing metastatic disease in breast cancer patients8,9,10.

Each TMEM doorway consists of three distinct types of cells: a perivascular macrophage, a tumor cell over-expressing the actin-regulatory protein mammalian enabled (Mena), and an endothelial cell, all in direct physical contact with each other1,5,9,10,11,12,13. The key event for the function of TMEM as an intravasation doorway is the localized release of vascular endothelial growth factor (VEGF) onto the underlying vessel by the perivascular macrophage14. VEGF can disrupt homotypic junctions between endothelial cells15,16,17,18,19, a phenomenon that results in transient vascular leakage, also known as "bursting" permeability as described in IVI studies 5. TMEM macrophages have been shown to express the tyrosine kinase receptor TIE2, which is required for VEGF-mediated TMEM function and homing of these macrophages to the perivascular niche5,20,21,22. In addition to regulating cancer cell dissemination and metastasis, TIE2+ macrophages have been shown to be central regulators of tumor angiogenesis21,22,23,24,25,26,27,28,29,30,31. As such, TIE2+ macrophages represent a critical constituent of the tumor microenvironment and the main regulator of the metastatic cascade.

To better conceptualize TMEM-mediated vascular permeability (i.e. "bursting"), it is very important to distinguish it from other modes of vascular permeability that are not associated with the dissolution of endothelial cell-cell junctions. In an intact endothelium (one whose tight and adherens junctions are not disrupted), there are three main types of vascular permeability: (a) pinocytosis, which may, or may not, be coupled to transcytosis of the ingested material; (b) transportation of material through endothelial fenestrae; and (c) transportation of material through the paracellular pathway, which is regulated by endothelial tight junctions15,16,17,18,19,32,33,34. Although deregulated in many tumors, the aforementioned modes of vascular permeability have been described mostly in the context of normal tissue physiology and homeostasis, the extremes of which are tissues with either limited permeability (e.g., blood-brain barrier, blood-testis barrier), or abundant permeability (e.g., fenestrated capillaries of the kidney glomerular apparatus)34,35,36,37.

Using multiphoton intravital imaging and multiplexed immunofluorescence microscopy, we are able to distinguish between TMEM-mediated vascular permeability ("bursting") and other modes of vascular permeability in breast tumors. To achieve this, we perform a single intravenous injection of a high-molecular weight, fluorescently-labeled probe in mice. Spontaneous bursting events can then be captured using intravital imaging in live mice; or alternatively, extravasation of the probe can be quantified by co-localization studies with blood vasculature (e.g. CD31+ or Endomucin+) and TMEM doorways using immunofluorescence microscopy. The protocols presented here describe both of these techniques, which could be used either independently or in conjunction with one another.

Protocol

All experiments using live animals must be conducted in accordance with animal use and care guidelines and regulations. The procedures described in this study were carried out in accordance with the National Institutes of Health regulations concerning the care and use of experimental animals and with the approval of the Albert Einstein College of Medicine Animal Care and Use Committee (IACUC).

1. Evaluation of "bursting permeability" using live animal imaging

  1. Transplantation of syngeneic breast tumors into mouse hosts with fluorescent macrophages
    1. Generate pieces of tumor tissue suitable for transplantation.
      1. Generate fluorescently-labeled tumors in mouse mammary cancer models by crossing the spontaneous, autochthonous, genetically engineered mouse mammary cancer model MMTV-PyMT mice with transgenic mice expressing fluorescent reporters38,39 [e.g., enhanced green fluorescent protein (EGFP), enhanced cyan fluorescent protein (ECFP), or Dendra2].
      2. Allow the MMTV-PyMT mice with fluorescently labeled tumors to grow to a size of no larger than 2 cm (approximately 10-12 weeks of age).
      3. Euthanize the tumor bearing MMTV-PyMT mice by placing them into a chamber with 5% isoflurane until 30 seconds after all respirations stop.
      4. Perform a cervical dislocation.
      5. Remove hair from the euthanized mouse’s abdomen using a topical depilatory cream.
      6. Place a Petri dish with DMEM/F12 cell culture medium on ice.
      7. Place the mouse and Petri dish on ice into the fume hood.
      8. Sanitize the mouse’s abdomen with 70% ethyl alcohol.
      9. Using sterile gloves and surgical tools (sterilized scissors, forceps, and blade) remove the tumors and place them into the Petri dish.
      10. Cut up the tumor into small pieces (~2 mm x 2 mm x 2mm in size), discarding any necrotic portions, while they are in the Petri dish.
    2. Transplant the tumor pieces into recipient hosts.
      1. Raise mice with genetically engineered fluorescently-labeled macrophages, e.g., MacGreen40 or MacBlue 41 mice (Csf1r-GAL4VP16/UAS-ECFP)
      2. Allow the FVB mice with fluorescently labeled macrophages to grow to an age of ~4-6 weeks).
      3. Anesthetize the FVB mice with fluorescently labeled macrophages in a chamber using 5% isoflurane with oxygen as a carrier gas.
      4. Reduce the anesthesia to ~3% isoflurane and apply ophthalmic ointment to the eyes of the mouse to prevent drying.
      5. Remove hair from over the 4th mammary gland of the mouse.
      6. Clean the skin with betadine. It is important to maintain sterile conditions throughout the rest of the procedure. This includes using sterilized instruments and reagents.
      7. Make a small incision ~2-3 mm just inferior to the 4th nipple.
      8. Dissect until the mammary fat pad is exposed.
      9. Take a tumor piece from the Petri dish and coat in artificial extracellular matrix.
      10. Transplant tumors underneath the 4th mammary fat pad.
      11. Close the incision using cyanoacrylate adhesive.
      12. Continuously monitor the animal until it fully recovers from anesthesia and is able to maintain sternal recumbency. Also, do not to return the animal to the company of other animals until full recovery.
      13. To minimize infections, add 1 mL of 100 mg/mL enrofloxacin antibiotic to the animal's drinking water bottle (8 fl oz).
      14. Allow tumors to grow until palpable (~5 mm; ~4 weeks).
      15. Depending on the experiment, allocate the mice into treatment groups, and perform the corresponding treatments, if applicable.
  2. Setup for intravital imaging      
    1. Turn on microscope's two-photon laser and detectors.
    2. Turn on the heating box and pre-heat the microscope's x-y stage.
    3. Place the custom-made stage insert42 into the x-y stage.
  3. Preparation of imaging window
    1. Prior to setting up for imaging, autoclave the custom-made circular imaging window frame42.
    2. Use a pipette or an insulin syringe to place a thin layer of cyanoacrylate adhesive to the window frame and affix in place an 8 mm circular cover glass.
      NOTE: 1) It is important to avoid getting residue on the clear aperture of the cover glass. 2) It is important to adhere the cover glass to the window frame at least 1 h before use for imaging.
    3. Carefully wipe clean any excess cyanoacrylate on the clear aperture of the cover glass using a laboratory wipe wetted with a small amount of acetone.
  4. Preparation of tail vein catheter for administration of fluids and fluorescent dyes during imaging
    1. Cut a 30 cm piece of polyethylene tubing to construct a tail vein catheter.
    2. Detach the needle portion of a 30 G needle from its Luer taper by gently bending the needle back and forth until it breaks.
      NOTE: The needle should be held close to the Luer taper and not the needle tip. This can be performed with pliers or forceps to grasp the needle in order to prevent needle stick injury.
    3. Insert the blunt end of the needle into one end of the polyethylene tubing.
    4. Insert the sharp end of another 30 G needle, keeping its Luer taper attached, to the opposite end of the tubing.
    5. Fill a 1 cc syringe with phosphate buffered saline, attach it to Luer taper of the assembled catheter, and flush the tail vein catheter making sure that there are no air bubbles in the system.
    6. For vascular labeling, fill a 1 cc syringe with 100 μL of 10 mg/kg 155 kDa dextran-tetramethylrhodamine (TMR) or quantum dots.
  5. Preparation of mouse for imaging
    1. Anesthetize the mouse in a cage underneath (~1 foot below) a heat lamp using 4%-5% isoflurane mixed with 100% oxygen set to a flow of 1.5-2 L per min.
    2. Turn on heat lamp over surgical working area. This step is critical for maintaining the core physiologic body temperature of the mouse during the surgery.
    3. Place the mouse under the heat lamp and lower the anesthesia to 2%-3% for the duration of the surgery.
      NOTE: Be sure to keep the heat lamp at a safe distance (~1 foot) away from the mouse to avoid overheating.
    4. Place ophthalmic ointment on the mouse’s eyes to prevent drying of the eyes and blindness.
    5. Test that the mouse is anesthetized by performing toe-pinch test. If animal withdraws, increase dosage of isoflurane by 1% and retest in 1-2 min.
    6. Insert the tail vein catheter into the most distal point on the tail possible.
    7. Affix the tail vein catheter to the tail with a small piece of lab tape that wraps around the tail and sticks to the needle to insure it does not get dislodged.
    8. Inject 50 μL per h of PBS through the tail vein catheter to provide adequate hydration. It is critical to avoid injecting too much fluid (no more than 200 μL per h) or any bubbles into the catheter as this can be fatal to the mouse.
    9. Remove hair on the abdomen over the 4th and 5th mammary glands using depilatory cream.
    10. Clean the skin with betadine and allow skin to dry.
    11. Make a longitudinal midline incision starting immediately superior to the genitals and carry the incision up to the level of the superior aspect of the 4th mammary gland.
    12. Carry the incision transverse to the superior aspect of the 4th mammary gland. It is critical to avoid compromising the blood supply at this point.
    13. Dissect the mammary fat pad off the peritoneum creating a tissue flap using sterile forceps and scissors.
      NOTE: Skin flap imaging is susceptible to significant motion artifacts and tissue dehydration. These are avoided, as described previously43,44, by affixing a rigid piece of rubber behind to the skin side of the flap (to stiffen the soft tissue and isolate it from the rest of the body) and then placing the tumor into a shallow imaging window to preserve the hydration. This is critical for stable imaging as the tumor and surrounding tissue in this setting are very compliant.
    14. Stabilize the skin flap by affixing (with cyanoacrylate glue) a small piece of rigid rubber measuring 2 cm x 2 cm to the skin side of the flap. The tumor should be in the center of the area being stabilized by the rubber.
    15. Keep exposed tissue hydrated with drops of PBS.
    16. Apply a small film of cyanoacrylate to the outer rim of the custom-made imaging window frame.
    17. Apply a small droplet of PBS (~10–20 μL) to the center of the cover glass.
    18. Dry the surrounding flap tissue with a laboratory wipe. It is critical to make sure that the cyanoacrylate on the window frame does not come into contact with the PBS on the glass, as this can cause the cyanoacrylate to polymerize and set prematurely.
    19. Affix the small imaging window to the tissue flap with the tumor at the center of the clear aperture.
    20. Remove the heating box from the stage.
    21. Transfer the anesthetized mouse and tail vein catheter to the microscope stage. Use extreme caution to ensure the tail vein catheter does not fall out.
    22. Place mouse on the stage in the prone position.
    23. Place the nose cone of isoflurane over the snout to ensure maintenance of anesthesia.
    24. Insert the window into the bore on the custom x-y stage plate.
    25. Place the heating box back onto the stage to maintain a physiological temperature.
    26. Monitor the animal's vital signs by attaching a pulse oximeter probe via clip sensor to the back paw.
    27. Slowly decrease isoflurane to 0.5%-1% to maintain adequate blood flow and avoid over anesthetizing the mouse.
  6. Intravital imaging
    NOTE: The imaging we describe in this section was performed on a custom-built two-laser multiphoton microscope that has been previously described5,39,45. Briefly, a femtosecond laser is used to generate 90 femtosecond pulsed laser light centered at 880 nm. Fluorescence light is detected with three of the four simultaneously acquiring detectors (Blue = 447/60, Green = 520/65, and Red 580/60; central wavelength/bandwidth) after separation from the excitation light by a dichroic (Chroma, Z720DCXXR). The microscope stand contains a 25x, 1.05 NA (numerical aperture) long working distance (2 mm) objective lens. It is important to note that, while we have used a custom-built microscope, the protocol described below can be accomplished on any commercially available multiphoton microscope as well.
    1. Place a drop of distilled water between the 25x, 1.05 NA microscope objective and the window’s cover glass to make optical contact.
    2. Use the microscope eyepiece to focus on areas with fluorescent tumor cells near to the surface of the window.
    3. Find flowing blood vessels and labeled macrophages. It is critical to have flowing blood vessels to assess the dynamics of the vasculature.
    4. Switch the microscope into multiphoton mode.
    5. Set the upper and lower limits of a z-series, which measures approximately 50 μm.
      1. Set the upper limit of the z-series by using the focus adjuster to move the objective to the desired start location, at the most superficial position, and marking this position as zero within the software by clicking on the Z position Top button.
      2. Set the lower limit of the z-series moving the objective to the deepest layer (typically 50-70 μm from the top for smaller tumors) and clicking the Z position Bottom button.
      3. Set the z-step size to 5 μm.
    6. Click the Time-Lapse panel button and set the time interval between acquisitions to at least 10 s to provide adequate time to replenish the water above the objective lens. This is done manually with a squeeze pipette on the objective during the long time lapse.
    7. Remove the syringe with PBS in the tail vein catheter and replace it with another syringe containing the 155 kDa dextran-TMR (tetramethyl rhodamine).
    8. Inject 100 μL of 155 kDa dextran-TMR via the tail vein catheter.
    9. After injection, replace the TMR syringe with the PBS syringe.
    10. Acquire a z-stack time-lapse imaging by clicking on the Z-Stack and Time-Lapse buttons, then clicking on the record button.
    11. Inject 50 µL of PBS every 30-45 min to maintain adequate hydration of the animal. Avoid injecting more than 200 μL at time as this can cause fluid overload.
  7. Euthanasia
    1. Increase the isoflurane to 5% and keep the animal under 5% isoflurane with nose cone in place until 30 s after respirations cease.
    2. Remove the mouse from the stage.
    3. Perform cervical dislocation.
  8. Image processing
    1. Load all images into ImageJ and format them into a 5 dimensional hyperstack (x, y, z, t, and color channel).
    2. Perform separation of spectral overlap (i.e.: GFP and CFP) and elimination of x-y drift from the hyperstacks using established methods38.
    3. Use the brightness and contrast adjustment to increase the white level of the blood channel so that the background signal becomes visible.
    4. For each z slice, carefully inspect each movie for signs of transient vascular leakage (a "burst"). Running the movies at fast frame rates (40 fps) may aid this identification.
    5. Once a burst has been identified, return the brightness for this channel to a normal level and crop the hyperstack to this region and z-slice.

2. Evaluation of extravascular dextran using fixed tissue analysis

  1. Tumor and sample preparation
    NOTE: The 2nd part of this protocol assumes that breast tumors have been harvested from an orthotopic transplantation mouse model of breast carcinoma (i.e. the MMTV-PyMT). This model could be the same as the one described in the 1st part of the protocol, although fluorescently-labeled tumors are not necessary at this point.
    1. Following the termination of the experimental pipeline (i.e. drug treatments, etc.), perform a tail-vain injection of 100 μL of 10 mg/mL 155 kDa dextran-tetramethyl rhodamine, 1 h before sacrificing the mice.
    2. Sacrifice the mice and harvest the breast tumors.
    3. Fix tumors in 10% formalin for 48-72 h and proceed to paraffin-embedding.
    4. Using a microtome, cut two 5 μm-thick sequential slides from the formalin-fixed paraffin-embedded (FFPE) tissues. One slide is used for staining the dextran, while the other will be used for performing TMEM triple-IHC, for reference.
      NOTE: The TMEM triple-immunohistochemistry protocol has been described elsewhere 10.
  2. IF staining and scanning for the first of the two sequential sections
    1. Submit slides to a standard de-paraffinization protocol. This includes two subsequent immersions in xylene (10 min each), followed by dehydration in serially diluted alcohol solutions (100%, 95%, 70%, and 50% EtOH in H2O for 2 min each immersion).
    2. Perform antigen retrieval by heating (close to boiling point) the sections submerged in citrate (pH 6.0-adjusted) for 20 min.
    3. Let the samples cool to room temperature for 15-20 min and then wash in PBS 3x for 2 min each wash.
    4. Block for 60-90 min in blocking buffer (10% FBS; 1% BSA; 0.0025% fish skin gelatin; 0.05% PBST, i.e. PBS with 0.05% Tween-20).
    5. Incubate samples with a mixture of primary rat and rabbit antibodies which target Endomucin and TMR, respectively, and then wash in PBST 3x, 2 min each.
    6. Incubate samples with a mixture of secondary donkey antibodies against rat IgG (conjugated to Alexa-647) and rabbit IgG (conjugated to Alexa-488), and then wash in PBST 3x 2 min each.
    7. Perform a routine DAPI staining (i.e. immersion in DAPI solution for 5-6 min), mount the slides using a glycerol-free "hard" mounting medium, and store in a dark place until scanning.
    8. For optimal results, scan the slides on a digital whole slide scanner.
  3. Image Analysis
    1. Capture 10 High Power Fields (HPFs) per case, using any software suitable for digital pathology.
    2. Save the Endomucin (Red) and TMR (Green) channels separately as TIFF.
    3. Using ImageJ, upload the TIFF files and convert them into 8-bit images.
    4. Threshold the 8-bit images to the level of the negative control, and generate two binarized images, showing the Endomucin and TMR "masks".
    5. From the Binary tools, select Fill Holes on the Endomucin mask.
    6. Generate and save the following five regions of interest: 1) The thresholded dextran image as "Dextran ROI" (ROI1), 2) the thresholded endomucin image as "Vascular ROI" (ROI2), 3) the inverted endomucin image as "Extravascular ROI" (ROI3), 4) the intersected "Extravascular ROI" and "Dextran ROI" image (ROI1 ∩ ROI3) as "Extravascular Dextran ROI" (ROI4), and 5) the entire image as "Tumor ROI" (ROI5).
    7. Divide the ROI4 area by the ROI5 area and multiply by 100 to generate the percent area that the extravascular dextran covers in the entire tumor.
    8. Repeat the process for 10-20 HPFs per case (depending on tissue availability) and generate an average Extravascular Dextran (% area) for each case.

Results

The experimental procedures described in this protocol article are briefly summarized and illustrated in Figure 1A-C.

To measure TMEM-mediated vascular permeability ("bursting activity") and to reduce experimental noise from other modes of vascular permeability (i.e. transcellular and paracellular, as explained in the introduction), we performed intravenous (i.v.) injection of high molecular weight probes, such as 155 kDa Dextran, conjugat...

Discussion

Here, we outline two protocols that can be applied to visualize and quantify a specific type of vascular permeability which is present at TMEM doorways and is associated with the disruption of vascular tight and adherens junctions. This type of vascular permeability is transient and controlled by the tripartite TMEM cell complex, as explained above5. The ability to identify and quantify TMEM-associated vascular permeability is crucial for the assessment of a pro-metastatic cancer cell microenviron...

Disclosures

The authors disclose no conflicts of interest.

Acknowledgements

We would like to thank the Analytical Imaging Facility (AIF) in the Albert Einstein College of Medicine for imaging support. This work was supported by grants from the NCI (P30CA013330, CA150344, CA 100324 and CA216248), the SIG 1S10OD019961-01, the Gruss-Lipper Biophotonics Center and its Integrated Imaging Program, and Montefiore’s Ruth L. Kirschstein T32 Training Grant of Surgeons for the Study of the Tumor Microenvironment (CA200561).

GSK co-wrote the manuscript, performed imaging for figure 1C and 3B, developed fixed tissue analysis protocol, and analyzed and interpreted all data; JMP co-wrote the manuscript, and performed the surgery and intravital imaging for Figure 1B,2C and 3A; LB & AC performed the surgery and intravital imaging for Figure 2B; RJ performed the surgery and intravital imaging for Figure 2A; JSC co-wrote the manuscript and analyzed and interpreted all data; MHO co-wrote the manuscript and analyzed and interpreted all data; and DE performed the surgery and intravital imaging for Figure 2D, co-wrote the manuscript, developed fixed tissue analysis and intravital imaging protocols, and analyzed and interpreted all data.

Materials

NameCompanyCatalog NumberComments
Anti-rabbit IgG (Alexa 488)Life Technologies CorporationA-11034
Anti-rat IgG (Alexa 647)Life Technologies CorporationA-21247
Bovine Serum AlbuminFisher ScientificBP1600-100
CitrateEng Scientific Inc9770
Cover Glass SlipsElectron Microscopy Sciences72296-08
Cyanoacrylate AdhesiveHenkel Adhesive1647358
DAPIPerkin ElmerFP1490
Dextran-Tetramethyl-RhodamineSigma AldrichT1287
DMEM/F12Gibco11320-033
Endomucin (primary antibody)Santa Cruz Biotechnologysc-65495
EnrofloxacinBayer84753076 v-06/2015
Fetal Bovine SerumSigma AldrichF2442
Fish Skin GelatinFisher ScientificG7765
Insulin SyringeBecton Dickinson309659
IsofluoraneHenry ScheinNDC 11695-6776-2
MatrigelCorningCB40234Artificial extracellular matrix
Needle (30 G)Becton Dickinson305128
Phosphate Buffered SalineLife Technologies CorporationPBS
Polyethylene TubingScientific Commodities IncBB31695-PE/1
Pulse OximeterKent ScientificMouseOx
Puralube Vet OintmentDechraNDC 17033-211-38
Quantum DotsLife Technologies CorporationQ21561MP
RubberMcMaster Carr1310N14
TMR (primary antibody)InvitrogenA6397
Tween-20MP BiologicalsTWEEN201
XyleneFisher Scientific184835

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