Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus

Summary

The choroid plexus (CP), an understudied tissue in neuroscience, plays a key role in health and disease of the central nervous system. This protocol describes a microdissection technique for isolating the CP and the use of scanning electron microscopy to obtain an overall view of its cellular structure.

Abstract

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in neuroscience. As it is becoming increasingly clear that this tiny structure plays a crucial role in health and disease of the central nervous system (CNS), it is of utmost importance to properly dissect the CP out of the brain ventricles in a way that allows downstream processing, ranging from functional to structural analysis. Here, isolation of the lateral and fourth brain ventricle mouse CP without the need for specialized tools or equipment is described. This isolation technique preserves the viability, function, and structure of cells within the CP. On account of its high vascularization, the CP can be visualized floating inside the ventricular cavities of the brain using a binocular microscope. However, transcardial perfusion required for downstream analysis can complicate the identification of the CP tissue. Depending on the further processing steps (e.g., RNA and protein analysis), this can be solved by visualizing the CP via transcardial perfusion with bromophenol blue. After isolation, the CP can be processed using several techniques, including RNA, protein, or single cell analysis, to gain further understanding on the function of this special brain structure. Here, scanning electron microscopy (SEM) on whole mount CP is used to get an overall view of the structure.

Introduction

Tight barriers separate the central nervous system (CNS) from the periphery, including the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier. These barriers protect the CNS against external insults and ensure a balanced and controlled microenvironment^1,2,3. While the BBB has been extensively studied over time, the blood-CSF barrier located at the choroid plexus (CP) has only gained increasing research interest during the last decade. This latter barrier can be found in the four ventricles of the brain (Figure 1A, B) and is characterized by a single layer of choroid plexus epithelial (CPE) cells surrounding a central stroma, leaky capillaries, fibroblasts, and a lymphoid and myeloid cell population (Figure 1C)^4,5,6. The CPE cells are firmly interconnected by tight junctions, thus preventing leakage from the underlying fenestrated blood capillaries into the CSF and brain. Additionally, transport across the CPE cells is regulated by a number of inward and outward transport systems that manage the influx of beneficial compounds (e.g., nutrients and hormones) from the blood to the CSF and the efflux of harmful molecules (e.g., metabolic waste, excess neurotransmitters) in the other direction^1,6. To be able to exert their active transport function, the CPE cells contain numerous mitochondria in their cytoplasm⁷. Moreover, the CP is the main source of CSF and acts as the gatekeeper of the brain by the presence of resident inflammatory cells¹. Due to its unique location between the blood and the brain, the CP is also perfectly positioned to carry out immune surveillance⁸.

Figure 1: Schematic overview of the location and composition of the choroid plexus (CP). (A,B) CP tissue is found within the two lateral, the third, and the fourth ventricles of (A) human and (B) mouse brains. (C) The CP tissue consists of a single layer of tightly connected cuboidal CP epithelium (CPE) cells surrounding fenestrated capillaries, loose connective tissue, and lymphoid and myeloid cells, and forms the blood-cerebrospinal fluid barrier (adapted and modified from reference²³). Figure created with Biorender.com. Please click here to view a larger version of this figure.

Over the past decade, increasing evidence, including several reports from our research group, have revealed that the CP plays a central role in health and disease^{9,10,11,12,13,14,15,16,17,18}. For example, it is known that the aging blood-CSF barrier displays morphological alterations in, among others, the nuclei, microvilli, and the basement membrane^1,19. Additionally, in the context of Alzheimer's disease, the overall barrier integrity is compromised and all of these age-related changes appear to be even more pronounced^1,8,20. In addition to morphological changes, the transcriptome, proteome, and secretome of the CP are altered during disease^12,21,22,23. Thus, advanced knowledge of the CP is essential to better understand its role in neurological diseases and potentially develop new therapeutic strategies.

An efficient method for accurate microdissection of the CP out of the brain ventricles is the first invaluable step to allow proper investigation of this tiny brain structure. On account of its highly vascularized nature (Figure 2B), the CP floating inside the ventricular cavities of the brain can be identified using a binocular microscope. However, transcardial perfusion is often required for downstream analysis, complicating the proper identification and isolation of the CP tissue (Figure 2C). If the further processing steps allow (e.g., in the case of RNA and protein analysis), the CP can be visualized via transcardial perfusion with bromophenol blue (Figure 2A). Several publications already describe the isolation of the CP from rat²⁴ and mouse pup brains²⁵. Here, a microdissection isolation technique is described to isolate the CP from adult mice. Importantly, this isolation technique preserves the viability, function, and structure of the cells within the CP. The isolation of the CP floating in the fourth and lateral ventricles is described here. In short, the mice are terminally anesthetized and, if necessary, transcardially perfused. However, it should be noted that perfusion can damage the structure of the cells within the CP. Consequently, if the sample is to be analyzed using transmission electron microscopy (TEM), serial block face scanning electron microscopy (SBF-SEM), or focused ion beam SEM (FIB-SEM), perfusion should not be performed. Next, the whole brain is isolated, and forceps are used to sagittally hemisect the brain. From here, the CPs floating in the lateral ventricles can be identified and dissected, while the CP from the fourth ventricle can be isolated from the cerebellar side of the brain.

Figure 2: Visualization of the (A-C) fourth and (D-F) lateral ventricle choroid plexus (CP) after (A,D) bromophenol blue perfusion, (B,E) no perfusion, and (C,F) perfusion with PBS/heparin. The images are taken with a stereo microscope (8x-32x magnification). Please click here to view a larger version of this figure.

Once the CP is properly dissected out of the brain ventricles, a whole repertoire of techniques can be applied to gain further understanding on the function of this structure. For example, flow cytometry or single cell RNA sequencing can be performed to quantify and phenotypically analyze the infiltrating inflammatory cells under certain disease conditions^26,27. In addition to the cellular composition, the molecular composition of the CP can be analyzed to assess the presence of cytokines and chemokines via enzyme-linked immunosorbent assay (ELISA), immunoblot, or through simultaneous analysis of multiple cytokines using the cytokine bead array²⁸. Moreover, transcriptome, vascular, immune cell histology, and secretome analyses can be performed on the microdissected CP explants²⁹. Here, scanning electron microscopy (SEM) on whole mount CP is used to obtain an overall view of the CP structure. SEM uses a beam of focused electrons to scan over the surface and create an image of the surface's topography and composition. Since the wavelength of electrons is much smaller than that of light, the resolution of SEM is in the nanometer range and superior to that of a light microscope. Consequently, morphological studies on the subcellular level can be performed via SEM. Briefly, the dissected CP is immediately transferred into a glutaraldehyde-containing fixative for an overnight fixation, followed by osmication and uranyl acetate staining. The samples are then treated with lead aspartate stain, dehydrated, and ultimately embedded for imaging.

Thus, this protocol facilitates the efficient isolation of the CP from the mouse brain ventricles, which can be further analyzed using a variety of downstream techniques to investigate its structure and function.

Protocol

All animal experiments described in this study were conducted according to the national (Belgian Law 14/08/1986 and 22/12/2003, Belgian Royal Decree 06/04/2010) and European legislation (EU Directives 2010/63/EU, 86/609/EEC). All experiments on mice and animal protocols were approved by the ethics committee of Ghent University (permit numbers LA1400091 and EC 2017-026).

1. Preparation

1. Anesthetics: Prepare a terminal anesthetic. For instance, a sodium pentobarbital (≥100 mg/kg) solution in phosphate-buffered saline (PBS) can be prepared.
2. Transcardial perfusion solution: Prepare 10 mL (per mouse) of PBS/heparin (containing 0.2% heparin) solution supplemented with 0.5% bromophenol blue.
   ​NOTE: If the downstream processing steps allow (e.g., in the case of RNA and protein analysis), the choroid plexus (CP) can be visualized via transcardial perfusion with bromophenol blue. If bromophenol blue is not compatible with the further processing steps (e.g., for imaging), use PBS/heparin to perfuse.
3. Prepare the solutions required for SEM analysis.
   1. Prepare 0.1 M Na-cacodylate buffer (pH 7.4). Prepare a solution of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer. Make 20 mL of this solution per sample.
   2. Prepare 2% osmium tetroxide in 0.1 M Na-cacodylate buffer (5 mL per sample).
   3. Prepare 50%, 70%, 85%, and 100% EtOH solutions. Prepare 5 mL of each EtOH solution per sample.

2. Microdissection of the choroid plexus out of the lateral and fourth ventricle

NOTE: Female, 9-week-old C57BL/6 mice were used in this study. However, the described isolation technique is independent of the strain, sex, and age of the adult mouse.

1. Isolate the mouse brain.
   1. Intraperitoneally inject a lethal dose of a short-acting barbiturate (>100 mg/kg, prepared in step 1.1) using a 26G needle to terminally anesthetize the mouse. Check the foot reflex of the mouse by pinching its rear paw with forceps.
   2. When there is no foot reflex, place the terminally anesthetized animal in the dorsal decubitus position and fix the mouse by pinning the limbs on a plate.
      NOTE: If transcardial perfusion of the mice is not needed, depending on the downstream analysis method (e.g., for SEM imaging), proceed to step 2.1.7. However, if perfusion is needed to remove blood cells or other components in the blood, the CP can be visualized as a blue structure floating in the brain ventricles via perfusion with bromophenol blue.
   3. Disinfect the chest by spraying 70% ethanol. Place a sterile drape around the surgical area. Make an incision of ~4 cm just under the diaphragm using a carbon steel surgical blade (see Table of Materials).
   4. Open the skin and expose the chest using surgical scissors. Cut the diaphragm completely open.
   5. Separate the thorax to expose the lungs and the beating heart.
   6. Transcardially perfuse the mice with 10 mL of the perfusion solution at a rate of 4.5 mL/min, using a perfusion pump (see Table of Materials). The perfusion will take ~2 min. Insert a 26G needle in the left ventricle to pump the solution into the systemic circuit. In addition, make a cut with surgical scissors in the right atrium so that blood can go out of the circulation.
      NOTE: A perfusion pump is preferred over manual administration of the fluid, as it will deliver the fluids at a precisely programmed rate and ensure that the shearing forces caused by the perfusion are not too strong. Excessive shearing forces will compromise the viability and structure of cells within the CP. Additionally, the perfusion flow rate recommended here is optimal to perfuse adult mice from 7 weeks onward. If younger mice are used, a lower flow rate should be used. It is also important to dab away the blood that comes out of the right atrium in order to preserve a clean surgical site.
   7. Decapitate the mouse.
      NOTE: Pay caution to cut off the head as low as possible to the shoulder to preserve the brain structure. If the cut is too high, the cerebellum can be damaged.
   8. After decapitation, cut open the scalp by a making an incision from between the ears to superior to the eyes. Pull the skin laterally to expose the skull. Then, cut open the skull by following the squamosal sutures, toward the nasal part.
      NOTE: It is important to gently remove the skull to maintain the brain integrity.
   9. Place the brain in an ice-cold Petri dish and add 1 mL of ice-cold 1x PBS on the brain tissue.
      NOTE:Coating of the dish is not needed.
2. Isolate the CP floating in the fourth ventricle.
   1. Gently cut off the cerebellum from the cerebrum with a scalpel. The cerebrum is the largest part of the brain, while the cerebellum is a much smaller part at the back of the brain. If necessary, remove remaining brain stem tissue parts from the cerebellum.
   2. Rotate the brain so that the cutting line is facing upward. Ensure that the fourth ventricle cavity is now visible in the middle of the section, with the CP floating at the dorsal site. If necessary, open up the ventricle a bit by pulling open the connective tissue with sharp forceps. In this way, the CP will be more visible and easier to reach.
   3. Gently tear the CP out of the ventricle wall using tiny sharp forceps.
      NOTE It is important to only touch and take out the CP in this step in order to not contaminate the sample with the surrounding tissue. Opening up the ventricles as described in step 2.2.2 will facilitate step 2.2.3.
3. Isolate the CP protruding out of the lateral ventricle.
   1. Use tiny sharp forceps to sagittally cut the brain into two hemispheres.
   2. Rotate the brain so that the cutting line is facing upward. To reveal the lateral ventricle, gently pull away the cortex from the thalamus.
   3. Retract the hippocampus to the midsagittal cutting line. The lateral ventricle is now visible with the CP lying at the bottom of the ventricle. If necessary, open up the ventricle a bit by pulling open the connective tissue with sharp forceps. In this way, the CP will be more visible and easier to reach.
   4. Use tiny sharp forceps to gently tear the CP out of the ventricle wall.
      ​NOTE: It is important to only touch and take out the CP in this step in order to not contaminate the sample with the surrounding tissue. Opening up the ventricles as described in step 2.3.4 will facilitate step 2.3.5. Use necessary caution in this step to preserve the structure of the CP as much as possible. It is easiest to start pulling the CP in the rostral to caudal direction.

3. Morphological analysis of CP tissue using scanning electron microscopy (SEM)

CAUTION: Toxic solutions are used in the following processing steps. It is recommended to perform the sample preparation in a fume hood.

1. Perform sample preparation for SEM as described below.
   1. Transfer the fresh isolated CP into freshly made fixation solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.4). Incubate overnight at 4 °C.
      NOTE: As the CP tissue is very fragile and thin, the tissue should be put in small specimen baskets (see Table of Materials) to transfer it between buffers. In this way, the structure of the tissue will be better preserved. A cacodylate-based fixative is used over a PBS-based one, as the strong cacodylate buffer can counter the low pH of glutaraldehyde in the EM fixative.
   2. Wash the sample 3x for 5 min each with 3-5 mL of 0.1 M Na-cacodylate buffer (pH 7.4).
   3. Post-fix the samples in 3-5 mL of 2% osmium tetroxide in 0.1 M Na-cacodylate buffer for 30 min. Wash the samples 3x for 5 min each with 3-5 mL of ultrapure water.
   4. Dehydrate the samples in a series of ice-cold solutions of increasing EtOH concentrations (50%, 70%, 85%, 100%), for 15 min per EtOH solution. Use a critical point dryer to properly point dry the sample.
      NOTE Changing from liquid to gas phase affects the surface tension, causing damage to the surface structure. The critical point drying step allows for the preservation of the surface structure.
   5. Position the sample carefully on a specimen mount provided with a carbon sticker (see Table of Materials).
   6. Coat the samples with a thin layer (2-5 nm) of platinum. To do so, mount a platinum source in a vacuum system between two high-current electrical terminals and heat the platinum to its evaporation temperature. A fine stream of platinum is deposited on the sample.
      NOTE: A more detailed protocol for this step is provided in reference³⁰. Besides platinum, gold or gold/palladium can also be used.
2. Visualize the CP samples with SEM (see Table of Materials). The visualization procedure of CP tissue via SEM is similar to that of other types of tissues and depends on the used software and instruments. A step by step SEM protocol with an accompanying video is available in reference³¹ or reference³⁰.

Representative Results

The described protocol facilitates the efficient isolation of the CP from the mouse brain lateral (Figure 2A-C) and fourth (Figure 2D-F) ventricles. After isolating the whole brain, forceps are used to sagittally hemisect the brain and identify the CPs floating in the lateral ventricles. The CP from the fourth ventricle can be isolated from the cerebellar side of the brain. Perfusion with bromophenol blue can be used to visualize the CP (Figure 2A,D); however, when bromophenol blue is not allowed in the further processing steps, perfusion with PBS/heparin (Figure 2C,F) or no perfusion (Figure 2B,E) can be performed. After isolating the CP out of the brain ventricles, a whole repertoire of analyses can be performed on the tissue. These include gene expression profiling (RT-qPCR)²⁸, cell type profiling (flow cytometry³², single cell RNA sequencing^26,27), and the detection of cytokines and chemokines²⁸ via enzyme-linked immunosorbent assay (ELISA), immunoblot, or multiplex immunoassays. Moreover, the microdissected CP can be placed in culture to make CP explants to further elucidate the function and, for example, study its secretome in response to a specific stimulus^23,28,29. In this manuscript, we describe how to perform scanning electron microscopy (SEM) to morphologically analyze the surface of choroid plexus epithelial (CPE) cells. Figure 3 shows overview images of the CP isolated from the fourth ventricle (Figure 3A) and the CP isolated from the lateral ventricle (Figure 3B), obtained via SEM. These images clearly show the typical C-shaped form of the lateral CP and the two-arm structure of the fourth ventricle CP.

Figure 3: Overview scanning electron microscopy (SEM) images of the dissected choroid plexus (CP) tissue. (A,B) Representative SEM image of the CP isolated out of the (A) fourth and (B) lateral ventricles of the mouse brain. Scale bar = 100 µm. Settings: electron high tension (EHT) = 5.00 kV; working distance (WD) = 4.8 mm Please click here to view a larger version of this figure.

By zooming in on the CPE cells, other cells on the apical side of the CPE cells can be detected (Figure 4A). The cell captured in the image is possibly an epiplexus cell, a macrophage-like cell that resides on the apical surface of the CP. However, it is not possible to identify the specific nature of this cell via SEM. In addition to SEM imaging, it is possible to perform two-photon imaging of the choroid plexus epithelium in live explants, as shown by Shipley et al.²⁹. While SEM facilitates the visualization of surface structures of the CP, two-photon imaging can enable the visualization of a broad range of cells and cellular processes as long as they can be fluorescently monitored. This includes visualization of vascular and immune cells, as well as the secretory events in the CP explant²⁹.

A higher SEM magnification revealed vesicle-like structures on the apical side of CPE cells (Figure 4B) as well as microvilli (Figure 4C). These microvilli protrude into the cerebrospinal fluid (CSF) and increase the CPE cell surface area between the cells and the CSF. These results show that the morphological alterations of the CPE cells in disease conditions can be investigated using SEM.

Figure 4: Detailed scanning electron microscopy (SEM) images of choroid plexus (CP) tissue. (A) A cell on the apical side of choroid plexus epithelial (CPE) cells. Scale bar = 2 µm. (B) Vesicle-like structures on the apical side of CPE cells. Scale bar = 10 µm. (C) Visualization of a CPE cell surface with its microvilli. Scale bar = 1 µm. Settings: electron high tension (EHT) = 5.00 kV; working distance (WD) = 4.9 mm; 3,000x magnification. Please click here to view a larger version of this figure.

Discussion

Here, a method to isolate the choroid plexus (CP) out of the lateral ventricle and the fourth ventricle of a mouse brain is described. This whole mounting method of the CP facilitates further analysis using a repertoire of techniques to get a complete view of the CP morphology, cellular composition, transcriptome, proteome, and secretome. Such analyses are crucial to gain a better understanding of this remarkable structure protruding from the ventricles of the brain. This knowledge is of immense research interest, as it is becoming increasingly clear that the CP plays a crucial role in health and disease^{9,10,11,12,13,14,15,16,17,18}.

Critical steps, modifications, and troubleshooting of the method
It is of utmost importance to check the foot reflex of the mouse to make sure that the terminal anesthesia is well performed. Besides ethical reasons, this also assures that the mouse is retained in the right place while performing the experimental procedure. The objective is to anesthetize the animal so that it does not experience pain during the procedure while the heart is beating at the time of perfusion. If further processing of the tissue requires the removal of blood, transcardial perfusion can be performed. Saline containing heparin is used here to avoid blood clotting. EDTA can also be used for this purpose, however, if it is necessary to preserve the cell viability, heparin is a better choice over EDTA. Perfusion should be started immediately when the heart stops beating due to excessive anesthesia. A perfusion pump is preferable over manual administration of the fluid, as this enables fluid delivery at a precisely programmed rate and ensures that the shearing forces caused by the perfusion are not too strong. Excessive shear forces will compromise the viability and structure of cells within the CP.

It takes a trained eye to be able to the see the CP floating in the mouse brain ventricles. For this reason, it is of utmost importance to practice a lot. It is recommended to start the training on mice perfused with bromophenol blue, as this will stain the CP in blue and make it easier to discriminate the tiny CP tissue from the rest of the brain (Figure 2A,D). In later trials, the CP can be isolated from a non-perfused mouse. This is harder compared to CP isolation from a bromophenol blue-perfused mouse, but the CP tissue can still be identified from its highly vascularized nature (Figure 2B,E). Only after extensive training it is possible to isolate the CP tissue out of the brain ventricles from a non-perfused mouse without contamination with brain parenchyma (Figure 2C,F).

The CP is a very fragile and thin structure, implying that its isolation should be performed with the necessary prudence in order to preserve the viability, function, and structure of cells within it. Thus, different steps in the described protocol need to be performed with the necessary precision and caution to preserve brain integrity. First, it is important to decapitate the mouse close to the shoulders. If the cut is too high, the cerebellum can be damaged, complicating the isolation of the CP in the fourth ventricle. Next, the skull needs to be removed carefully to not damage the brain. Once the brain is isolated, it is crucial to keep it cold to prevent the brain from getting mushy, as this will significantly complicate CP isolation. To do so, it is important to place the brain on an ice-cold Petri dish and add ice cold PBS over the brain. A certain level of training is necessary to perform the described isolation technique as this will significantly shorten the processing time between the decapitation of the mouse and the final isolation of the CP. A short isolation process will enhance the viability and structure preservation of the isolated tissue. Finally, the tools used for isolation, especially the forceps, need to be sharp and pointed to facilitate the fast and efficient isolation of the CP out of the ventricles. However, care should be taken to not damage the integrity of the structure during isolation.

To preserve tissue integrity during sample processing for SEM, the CP can be put in small specimen baskets when transferring between buffers, so that touching of the tissue itself can be avoided. Moreover, it is recommended to perform critical point drying when moving the tissue out of the EtOH solutions. This ensures the preservation of surface structure of the CP, including microvilli. Surface tension, caused by the transition from liquid to gas phase, could damage such structures.

Limitations of the method
As mentioned earlier, the CP is a tiny structure, and depending on the downstream analyses (e.g., flow cytometry), it may be necessary to pool the CP from different mice. A significant hurdle in CP isolation is the identification of the structure when it is still floating in the ventricle. The highly vascularized nature of the CP facilitates its correct identification (after some training) inside the ventricular cavities of the brain. However, if blood-containing components need to be removed for further analysis, transcardial perfusion is essential. Depending on the downstream analysis, perfusion with bromophenol blue can be performed, which will stain the CP blue, thereby facilitating the isolation of the tissue. Unfortunately, this visualization technique is not always possible (e.g., for immunohistochemistry staining). In such cases, the CP needs to be isolated blindly, which requires adequate training. Playing with the light diffraction of the microscope can help to identify the CP floating in the ventricles. In addition, this manuscript describes the isolation of the CP floating in the fourth and lateral ventricles, while isolation of the third ventricle CP is not discussed.

Significance with respect to existing methods
In this protocol, the CP is first dissected out of the brain ventricles before further analysis is performed. If a staining procedure is used as follow-up read-out, it is also possible to stain brain sections that contain the CP. The added value of this latter technique is that the orientation of the CP tissue within the ventricles and the brain is still visible, which is not the case if the CP is first microdissected out of the brain ventricles. On the other hand, staining a brain section containing CP provides information only from that specific section, whereas staining the isolated CP can help gather information about the whole CP.

The advantage of the described technique is that it preserves the viability, function, and structure of the cells within the CP. Moreover, this method facilitates the complete isolation of the CP floating in the fourth and lateral brain ventricles in adult mice. Methods for isolation of the CP (e.g., from rat brains²⁴ and mouse pup brains²⁵) have been previously reported. However, it is important to consider that the isolation technique can differ from species to species and between young and adult ages.

Additional applications of the technique
Besides preserving the viability, function, and structure of cells within the CP, this isolation technique facilitates the application of downstream techniques to gain a deeper understanding of CP function. For example, flow cytometry or single cell RNA sequencing can be performed to quantify and phenotypically analyze CP cells and infiltrating cells under certain disease conditions^26,27. In addition, the molecular composition of the CP can be investigated to assess the presence of cytokines and chemokines via enzyme-linked immunosorbent assay (ELISA), immunoblot, or simultaneous analyses of multiple cytokines by a so-called cytokine bead array. Moreover, transcriptome, vascular, immune cell histology, and secretome analyses can be performed on the microdissected CP explants²⁹.

Materials

Name	Company	Catalog Number	Comments
26G x 1/2 needle	Henke Sass Wolf	4710004512
Aluminium specimen mounts	EM Sciences	75220
Cacodylate buffer	EM Sciences	11652
Carbon steel surgial blades	Swann-Morton	0210	size: 0.45 mm x 12 mm
Carbon adhesive tabs -12 mm	EM Sciences	77825-12
Critical point dryer	Bal-Tec	CPD030
Crossbeam 540	Zeiss		SEM system
Forceps	Fine Science Tools GmbH	91197-00
Glutaraldehyde	EM Sciences	16220
Heparin	Sigma-Aldrich	H-3125
Ismatec Reglo ICC Digital Peristaltic pump 2-channel	Metrohm Belgium N.V	CPA-7800160
Osmium Tetroxide	EM Sciences	19170
Paraformaldehyde	Sigma-Aldrich	P6148
Phosphate buffered saline (PBS)	Lonza	BE17-516F
Platinum	Quorum	Q150T ES	PBS without Ca++ Mg++ or phenol red; sterile filtered
Sodium pentobarbital	Kela NV	514
Specimen Basket Stainless Steel	EM Sciences	70190-01
Stemi DV4 Stereo microscope	Zeiss
Surgical scissors	Fine Science Tools GmbH	91460-11