Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Podsumowanie

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

Streszczenie

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

Wprowadzenie

Taste buds are collections of 50-100 specialized epithelial cells that bind subsets of chemical-taste stimuli present in the oral cavity. Taste-transducing cells are generally thought to exist as types^{1,2,3,4,5,6,7,8,9}, initially based on electron microscopy criteria that were later correlated with molecular markers. Type II cells express phospholipase C-beta 2 (PLCβ2)² and transient receptor potential cation channel, subfamily M member 5¹ and include cells that transduce sweet, bitter, and umami^1,10. Type III cells express carbonic anhydrase 4 (Car4)¹¹ and synaptosomal-associated protein 25⁸ and denote cells that primarily respond to sour taste¹¹. The cells that transduce saltiness have not been as clearly delineated^12,13,14, but could potentially include Type I, Type II and Type III cells^{15,16,17,18,19}.The taste-bud environment is complex and dynamic, given that taste-transducing cells continuously turn over throughout adulthood and are replaced by basal progenitors^3,20,21. These taste-transducing cells connect to pseudo-unipolar nerve fibers from the geniculate and petrosal ganglia, which pass taste information to the brainstem. These neurons have primarily been categorized based on the kind of taste information they carry^22,23 because information about their morphology has been elusive until recently²⁴. Type II cells communicate with nerve fibers via calcium homeostasis modulator protein 1 ion channels²⁵, whereas Type III cells communicate via classical synapses^8,26. Further characterization of taste bud cells-including transducing cell type lineages, factors that influence their differentiation, and the structures of connecting arbors are all areas of active investigation.

Taste-bud studies have been hindered by several technical challenges. The heterogenous and dense tissues that make up the tongue significantly reduce antibody permeability for immunohistochemistry^27,28,29. These obstacles have necessitated sectioning protocols that result in the splitting of taste buds across sections so that measurements are either approximated based on representative sections or summed across sections. Previously, representative thin sections have been used to approximate both volume values and transducing-cell counts³⁰. Thicker serial sectioning allows for the imaging of all taste-bud sections and the summing of measurements from each section³¹. Cutting such thick sections and selecting only whole taste buds biases sampling towards smaller taste buds^32,33,34. Nerve innervation estimates from sectioned taste buds have been based on analyses of pixel numbers^13,35, if quantified at all^36,37,38. These measurements completely ignore the structure and number of individual nerve arbors, because arbors are split (and usually poorly labeled). Lastly, although peeling away the epithelium does permit entire taste buds to be stained^39,40, it also removes taste-bud nerve fibers and could disrupt the normal relationships between cells. Therefore, investigations of the structural relationships within taste buds have been limited because of this disruption caused by staining approaches.

Whole-structure collection eliminates the need for representative sections and allows the determination of absolute-value measurements of volumes, cell counts, and structure morphologies⁴¹. This approach also increases accuracy, limits bias, and reduces technical variability. This last element is important because taste buds show considerable biological variability both within^34,42 and across regions^43,44, and whole taste-bud analyses allow absolute cell numbers to be compared between control and experimental conditions. Furthermore, the ability to collect intact taste buds permits the analysis of the physical relationships between different transducing cells and their associated nerve fibers. Because taste-transducing cells may communicate with each other⁴⁵ and do communicate with nerve fibers⁴⁶, these relationships are important for normal function. Thus, loss-of-function conditions may not be due to a loss of cells, but instead to changes in cell relationships. Provided here is a method for collecting whole taste buds to achieve the benefits of absolute measurements for refining volume analyses for both taste buds and their innervations, taste-cell counts and shapes, and for facilitating analyses of transducing-cell relationships and nerve-arbor morphologies. Two workflows are also presented downstream of this novel whole-mount method for tissue preparation: 1) for analyzing taste bud volume and total innervation and 2) for sparse-cell genetic labeling of taste neurons (with subsets of taste-transducing cells labeled) and subsequent analyses of taste-nerve arbor morphology, numbers of taste-cell types and their shapes, and the use of image analysis software to analyze the physical relationships between transducing cells and those between transducing cells and their nerve arbors. Together, these workflows provide a novel approach to tissue preparation and for the analyses of whole taste buds and the complete morphology of their innervating arbors.

Protokół

NOTE: All animals were cared for in accordance with the guidelines set by the U.S. Public Health Service Policy on the Humane Care and Use of Laboratory Animals and the NIH Guide for the Care and Use of Laboratory Animals. Phox2b-Cre mice (MMRRC strain 034613-UCD, NP91Gsat/Mmcd) or TrkB^CreER mice (Ntrk2^{tm3.1(cre/ERT2)Ddg}) were bred with tdTomato reporter mice (Ai14). Advillin^CreER47 were bred with Phox2b-flpo⁴⁸ and Ai65. For 5-ethynyl-2′-deoxyuridine (EdU) injections, the EdU was prepared and doses calculated according to Perea-Martinez et al.⁴⁹.

1. Preparation of materials

1. Preparation of solutions
   1. Dissolve 5.244 g of monobasic sodium phosphate and 23.004 g of dibasic sodium phosphate in double-distilled water (ddH₂O) on a stir plate. Adjust the pH to 7.4, and bring the total volume to obtain 1 L of 0.2 M sodium phosphate buffer (PB).
   2. Dissolve paraformaldehyde in ddH₂O in a fume hood by heating while stirring on a stir plate until the solution reaches 90 °C. Add 4 M NaOH solution dropwise to clear the paraformaldehyde, and filter the solution using a vacuum Erlenmeyer flask and ceramic filter with filter paper. Add an equal volume of 0.2 M PB, and adjust the pH to 7.4 to obtain 4% PFA in 0.1 M PB.

2. Tissue preparation

1. Tissue collection
   1. Sacrifice mice using an anesthetic overdose with a working solution containing 10 mL of sterile saline and 0.25 mL of a stock solution containing 5 g of 2,2,2-tribromoethanol and 5 mL of tert-amyl alcohol. Perfuse transcardially with 4% PFA in 0.1 M PB; remove the tongues and the palate.
   2. Isolate the circumvallate taste buds using a coronal cut separating the posterior tongue, behind the intermolar eminence; cut off the taste buds with a razorblade; and then bisect the anterior tongue at the midline. Post-fix overnight at 4 °C with 4% PFA in 0.1 M PB.
   3. Cryoprotect the tissue in 30% sucrose overnight at 4 °C.
      ​NOTE: The tissue can be frozen in optimal cutting temperature (OCT) compound using 2-methylbutane chilled in a beaker on dry ice and stored at -80 °C if the procedure has to be paused here.
2. Fungiform taste buds
   1. Chill 2-methylbutane in a beaker on dry ice in preparation for step 2.2.8.
   2. Thaw and rinse the tongue in 0.1 M PB. Place one half of the anterior tongue containing the fungiform papillae on a glass slide under a dissecting microscope.
   3. Use blunt-ended forceps and dissection scissors to remove the muscle. Use blunt-ended forceps to hold the tissue open as the lingual epithelium is curved, and ensure a flat orientation by keeping the blades of the coarse dissection scissors parallel to the epithelium.
   4. Discard the ventral non-keratinized epithelium of the tongue as it contains no taste buds.
   5. Use fine dissection scissors for closer dissection to the underside of the keratinized epithelium.
      ​NOTE: It is important to dissect close to the epithelium so that the remaining muscle is of uniform thickness, and the surface is smooth to ensure uniform antibody penetration. The consequence of non-uniform thickness of the remaining muscle will be uneven sectioning on the cryostat, with exposure of the epithelium in areas with less muscle and a thicker layer of muscle for other areas, which impedes antibody penetration.
   6. Use the blunt-ended forceps to lay a piece of epithelium into a tissue mold (muscle side down), and ensure that it lays flat. Once the tissue is flat, add a drop of OCT to the tissue.
      ​NOTE: Given that the tip of the tongue is curved, it may be necessary to make a cut in the epithelium where it is curved so that the tissue can be made to lay flat.
   7. Place the tissue mold on a metal base (previously cooled in dry ice) under the dissecting scope. Continue to tap the tissue lightly with the forceps until the OCT has frozen to ensure the tissue freezes as flat as possible.
   8. Once the OCT has frozen, quickly add additional OCT, and place the mold in a beaker of 2-methylbutane (cooled in dry ice) until frozen.
   9. Cryostat sectioning
      NOTE: The cryostat is used for fine removal of remaining subcutaneous tissue, which may inhibit antibody penetration (Figure 1).
      1. Mount the OCT molds on the cryostat, and cut 20 µm sections. Collect each section, and view it under the light microscope to assess its proximity to the base of the epithelium (Figure 1E - H).
      2. After the tissue is shaved from the underside of the epithelium, thaw the epithelium and rinse it twice in 0.1 M PB on a shaker.
3. Circumvallate taste buds
   1. Using a coronal cut with a razorblade, separate the circumvallate papilla from the anterior tongue. Use two parasagittal cuts with the same razorblade to remove the tissue lateral to the papilla under a dissecting scope. Place the papilla in a tissue mold using forceps so that one lateral edge of the circumvallate papilla faces the bottom of the tissue mold.
      NOTE: The tissue can be frozen in OCT using 2-methylbutane chilled in a beaker on dry ice and stored at -80 °C if the procedure has to be paused here.
   2. Cut the tissue into 90 μm floating sections on the cryostat.
4. Taste buds on the palate
   1. Cut the hard palate anterior to the junction of the soft and hard palate (Figure 2). Use scissors to separate the soft palate from the underlying tissue, making sure any remaining bone fragments are cut away. Remove additional muscle and connective tissue.
      NOTE: Once removed, all tissue that remains will consist of glands and loose connective tissue, which are lightly adhered to the underside of the palate.
   2. Hold the palate with blunt-ended forceps, and remove the remaining glands and loose connective tissue by gently scraping them with a razor blade.
      NOTE: The tissue can be frozen in OCT using 2-methylbutane chilled in a beaker on dry ice and stored at -80 °C if the procedure has to be paused here.

3. Immunohistochemistry staining

1. Wash the tissues with 0.1 M PB, 3 x 15 min. Place the tissues in 1 mL tubes with blocking solution (3% donkey serum, 0.5% non-ionic surfactant (see the Table of Materials), 0.1 M PB) at 4 °C overnight.
2. Remove the blocking solution, and incubate the tissue in primary antibody (rabbit anti-PCLβ2) in antibody solution (0.1 M PB, 0.5% non-ionic surfactant) for 5-days at 4 °C.
3. Wash with 0.1 M PB, 4 x 15 min each wash, and incubate in secondary donkey anti-rabbit 488 antibody (1:500) in antibody solution for 2 days at 4 °C.
4. Wash with 0.1 M PB, 4 x 15 min each wash, and block with 5% normal rabbit serum in antibody solution.
5. Wash with 0.1 M PB, 4 x 15 min. Incubate with donkey anti-rabbit blocking antibody (20 µg/mL) in antibody solution for 2 days at 4 °C.
6. Wash with 0.1 M PB, 4 x 15 min each wash, and then incubate with primary antibody dsRed (rabbit) conjugated to a fluorescent label (according to the manufacturer's instructions) in an antibody solution for 5 days at 4 °C.
7. Wash with 0.1 M PB, 4 x 15 min each wash, and then incubate with primary antibody (goat anti-Car4 (1:500)) in antibody solution for 5 days at 4 °C.
8. Wash with 0.1 M PB, 4 x 15 min each wash. Incubate with secondary donkey anti-goat 647 antibody (1:500) in antibody solution for 2 days at 4 °C.
9. Wash with 0.1 M PB, 4 x 15 min each wash, and mount (epithelial side up) in aqueous mounting media, and place a coverslip over the tissue section.
   ​NOTE: If using antibodies from different species, as in the case with keratin-8 and dsRed only, add all primary antibodies to the antibody solution in step 3.2 and all secondary antibodies in step 3.3 before proceeding to step 3.9.

4. Confocal imaging and deconvolution

1. Capture confocal images using a confocal microscope with a 60x objective (Numerical Aperture= 1.40), 4 ms/pixel, zoom of 3, Kalman of 2, and size of 1024 x 1024. Select a step size of 0.47 mm along the z-axis. For capturing innervation to the papilla, use a zoom of 2.5 if the field of view with a zoom of 3 is too narrow to capture all the innervation to the papilla.
2. To deconvolute the images, note that some settings will automatically import with the image; therefore, fill in the remaining details for modality, objective lens, numerical aperture, immersion medium, sample medium, and fluorophores captured in the image. Then, select 3D Deconvolution.

5. Image analysis

1. Taste bud and innervation volume
   1. Import deconvoluted image stacks to a pixel-based image analysis software (see the Table of Materials) to determine the taste bud volume and volume of total innervation within the taste bud.
      1. Uncheck Volume in the main Object menu.
      2. Select Add new surfaces from the Object menu. Select Skip automatic creation, edit manually, and then select Contour.
      3. Click on Select and observe the arrow appearing as a + to help trace the border of the taste bud. Move the slicer and outline the taste bud in each optical section. Once the contours are complete, click on Create Surface.
      4. Observe the taste-bud volume object now appearing in the main Object menu. Locate the volume of the taste bud under Tools.
   2. Volume of innervation within the taste bud
      1. Select the pencil icon under the taste bud object, then select Mask All.
      2. In the dropdown menu, select the fluorescent channel that corresponds with the nerve fiber label. Check Create Duplicate Channel.
      3. Check Set voxels outside surface to:, and type 0 in the box.
      4. Observe the new channel appearing in the Display Adjustment window, which is an unaltered duplicate of the fluorescent channel selected within the taste bud.
      5. In the main Object menu, select Create New Surface. Uncheck Skip automatic creation, edit manually.
      6. Click twice on the blue arrow to proceed to the next step.
      7. Click on Delete, then click on the green double arrow to complete the surface, which represents the volume of the nerve fibers present within the taste bud. To find the value for the volume, select Tools under the nerve fiber Object menu, and select Volume from the dropdown menu.
   3. Volume of innervation to the papilla
      1. Create a volume of the taste bud as described in section 5.1.
      2. Select the pencil icon under the taste-bud object, then select Mask All.
      3. In the dropdown menu, select the fluorescent channel that corresponds with the nerve fiber label. Check Create Duplicate Channel.
      4. Check Set voxels inside surface to: and type 0 in the box. Click OK.
      5. Generate a surface by clicking on Add New Surface. Select Segment only a Region of Interest.
      6. Click on the blue arrow, and increase the Z-value so that the region of interest begins at the base of the taste bud.
      7. Click twice on the blue arrow to proceed to the next step.
      8. Click on Delete, then click on the green double arrow to complete the surface, which represents the volume of the innervation to the papilla. To find the value for the volume, select Tools under the nerve fiber Object menu, and select Volume from the dropdown menu.
   4. Terminal arbor contact analysis
      1. Image preparation
         1. Go to the Edit menu and select Crop 3D. Crop the image on all sides, removing space outside of the taste bud.
            NOTE: Crop as close to the taste bud without removing any relevant structure-any excess image will lengthen processing time.
         2. Select Edit from the main menu, click Change data type. Select To: 32 bit float from the dropdown menu.
         3. Select Add new surfaces from the Object menu. Click on Skip automatic creation, edit manually, select Contour, and then drag the slice position to the right to find the total number of optical slices.
         4. Generate isometric voxels, and make sure that instead of the voxels being rectangles (0.0691 x 0.0691 x 0.474) as XxYxZ, respectively, the voxels are 0.0691 x 0.0691 x 0.0691 by dividing the rectangles into cubes with identical values for fluorescence intensity as the original, rectangular voxel. Select Image Properties. Then, divide the Z value for Voxel Size by the X or Y value (= 0.474/0.0691), and multiply that value by the number of slices (optical sections) found in the previous step.
         5. Go back to Edit on the main menu, and select Resample 3D.
         6. Replace the Z value (number of slices) with the newly calculated value.
      2. Creating automatic surfaces based on fluorescence
         1. Click on Add New Surface again to add a new surface, deselect Segment only a region of interest, and click on Next.
         2. From the Source channel dropdown menu, select the channel for one of the taste-transducing cell types. Unselect Smooth and continue to the next step.
         3. Do not alter anything on the next screen that shows the range of fluorescent intensities present in the image. Click on the blue arrow at the bottom again to move to the next step.
         4. Click on Delete, then click on the green double arrow to complete the surface.
         5. Go to the Object menu on the left hand of the screen where the completed cell surface will appear and will be called something generic such as Surface 2. Double click on the surface name to name it according to what the label represents.
            NOTE: In this case, the surface generated is based on the PLCß2-labeled cell.
         6. If there are dots instead of a surface, under the menu in the lower left-hand corner, click on Surface instead of Center point. Then, click OK when prompted.
         7. Repeat steps 5.3.2.1-5.3.2.5 for the other taste-transducing cell markers and the nerve fiber marker.
         8. Save (export) the progress.
         9. Click on a cell type Surface in the main menu. From that object's menu, click on Tools, then click on Distance Transformation.
         10. Wait for a pop-up box entitled XTDistanceTransformation to appear. Select Outside Surface Object. Make note of the new channel that appears in the Display Adjustment menu called Distance to surface name.
         11. Select the Nerve Fiber surface from the Object menu. Click on the pencil icon and then Mask All.
         12. From the dropdown menu that appears, select the new channel Distance to surface name. Make note of the new channel that appears in the Display Adjustment window called Masked Distance to surface name.
         13. Create a new object using the main Object menu. Unselect Segment only a region of interest and click on the blue arrow. Select the Masked Distance to PLCß2 channel and uncheck Smooth.
         14. On the next screen, check if there are any regions where the taste-transducing cells are within the smallest distance from the nerve fibers discernable by this software. To do this, set a limit of 0.01-0.11 µm to check for any receptor cell fluorescence this close to the nerve fibers.
         15. Type 0.01 in the green box on the left side to set the lower threshold, press Tab. Then click on the red button and type 0.11 to set the upper threshold. Press Tab and then click on the blue arrow at the bottom to move on to the next step.
         16. Click on Delete and then the green double arrow to finish.
         17. Rename this surface Within 0.01-0.11 of PLCß2 in the Object menu. Select Within 0.01 - 0.11 of PLCß2 surface in the Object menu.
         18. Select the pencil then click on Mask All. Select the red (nerve fiber) channel from the dropdown menu and click OK. Make note of the new channel that appears in the Display Adjustment window called Masked CHS2.
         19. Click on the name of the channel; rename the channel Within 0.01 - 0.11 of PLCß2.
             NOTE: This is a fluorescent channel that represents a duplicate of the red fluorescent channel present within the surface created.
         20. Click on white in the middle of the color selector. Select a color that contrasts with the colors of the structures.
         21. Export (i.e., save) the file at this stage.
         22. Repeat steps 5.4.2.9-5.4.2.21 for other taste-transducing cell markers.
         23. Export (i.e., save) the file at this stage.
             NOTE: To analyze the proximity of one labeled cell type to another, simply replace the Nerve Fiber surface in step 5.4.2.11 (and the following steps) with the object of interest and the equivalent components that pertain to each subsequent steps.

6. Neuron arbor reconstruction and absolute cell number quantification

1. Terminal arbor tracing and analysis
   1. Open the deconvoluted image file in a 3D vector-based image analysis software (see the Table of Materials), select Trace, click on Neuron, and then click on Dendrite.
   2. Scroll to the base of the taste bud in the image stack. Trace each fiber to the end while scrolling through the image stack.
   3. When at the branch end, right click on the end and select Ending. At branch points, right click and select Bifurcating Node.
      NOTE: This enables tracing one branch to the end and then returning to the bifurcating point and tracing the other branch with the program recognizing that this tracing is still of the same neuron.
   4. Save the data file as a .DAT file, which can then be opened for analysis in the 3D vector-based image analysis software.

7. Cell number quantification

1. Quantify the labeled taste bud cells in any image analysis software package as long as distinct markers for transducing cell types anchored to the z-position can be placed at the nuclear level.

Wyniki

Staining of the lingual epithelium with antibodies to dsRed and keratin-8 (a general taste-bud marker) labeled both whole taste buds and all taste-bud innervation in Phox2b-Cre:tdTomato mice^50,51 (Figure 3A). Imaging these taste buds from their pores to their bases gave the highest resolution x-y plane images (Figure 3A,B). The contour function of the pixel-based imaging program was used...

Dyskusje

The development of an approach to consistently collect and stain whole taste buds from three oral cavity taste regions (fungiform, circumvallate, and the palate) provides significant improvements for analyzing taste-transducing cells, tracking newly incorporated cells, innervation, and relationships between these structures. In addition, it facilitates the localization of a potential secondary neuron marker both within or outside of a labeled population⁵⁰. This is particularly relevant given that ...

Materiały

Name	Company	Catalog Number	Comments
2,2,2-Tribromoethanol	ACROS Organics	AC421430100
2-Methylbutane	ACROS	126470025
AffiniPure Fab Fragment Donkey Anti-Rabbit IgG	Jackson ImmunoResearch	711-007-003	15.5μL/mL
Alexa Fluor® 647 AffiniPure Donkey Anti-Rat IgG	Jackson Immuno Research	712-605-150	(1:500)
AutoQuant X3 software	Media Cybernetics
Blunt End Forceps	Fine Science Tools	FST 91100-12
Click-iT™ Plus EdU Cell Proliferation Kit	Molecular Probes	C10637	Follow kit instructions
Coverglass	Marienfeld	107242
Cytokeratin-8	Developmental Studies Hybridoma Bank (DSHB), (RRID: AB_531826)	Troma1 supernatant	(1:50, store at 4°C)
Dissection Scissors (coarse)	Roboz	RS-5619
Dissection Scissors (fine)	Moria	MC19B
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	ThermoFisher Scientific	A21206	(1:500)
Donkey anti-Rabbit, Alexa Fluor® 555	ThermoFisher Scientific	A31572	(1:500)
DyLight™ 405 AffiniPure Fab Fragment Bovine Anti-Goat IgG	Jackson Immuno Research	805-477-008	(1:500)
Fluoromount G	Southern Biotech	0100-01
Glass slides	Fisher Scientific (Superfrost Plus Miscroscope Slides)	12-550-15
Goat anti-Car4	R&D Systems	AF2414	(1:500)
Imaris	Bitplane	pixel-based image analysis software
Neurolucida 360 + Explorer	MBF Biosciences	3D vector based image analysis software
Normal Donkey Serum	Jackson Immuno Research	017-000-121
Normal Rabbit Serum	Equitech-Bio, Inc	SR30
Olympus FV1000		(multi-Argon laser with wavelengths 458, 488, 515 and additional HeNe lasers emitting 543 and 633)
Paraformaldehyde	EMD	PX0055-3	4% in 0.1M PB
Rabbit anti-dsRed	Living Colors DsRed Polyclonal Antibody; Clontech Clontech Laboratories, Inc. (632496)	632496	(1:500)
Rabbit anti-PLCβ2	Santa Cruz Biotechnology	Cat# sc-206	(1:500)
Sodium Phosphate Dibasic Anhydrous	Fisher Scientific	BP332-500
Sodium Phosphate Monobasic	Fisher Scientific	BP330-500
tert-Amyl alcohol	Aldrich Chemical Company	8.06193
Tissue Molds	Electron Microscopy Sciences	70180
Tissue-Tek® O.C.T. Compound	Sakura	4583
Triton X-100	BIO-RAD	#161-0407
Zenon™ Alexa Fluor™ 555 Rabbit IgG Labeling Kit	ThermoFisher Scientific	Z25305	Follow kit instructions