We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.
Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.
Three dimensional (3D) culture models that more accurately mimic the tumor architecture and microenvironment in vivo are important for studies aimed to dissect the complex interactions between cells and their microenvironment and to test the efficacy of candidate therapies. Tumor dimensionality impacts oxygen and nutrient gradients, the uniformity of drug exposure, interstitial pressure/blood flow, and 3D architecture1-4. The presence of an appropriate stromal microenvironment contributes to tumor dimensionality and influences cell-ECM signaling and paracrine signaling between stromal cells and malignant epithelial cells. The effects of tumor dimensionality and the microenvironment on cellular function are well established, with both factors altering drug response1,3,5-8. Additionally, cellular growth kinetics, metabolic rates, and cell signaling differ between two dimensional (2D) culture and culture in 3D, with these factors affecting cellular response1,3,8-10.
In vitro, the tumor surrogate microenvironment can be modulated by including representative ECM constituents and stromal cell populations. Malignant epithelial cells are influenced by the ECM and cancer-associated stromal cells either in a synergistic/protective manner to promote tumor progression or in a suppressive manner to inhibit further tumor propagation5,6,10. In either context, the stroma can affect therapeutic response and drug delivery via paracrine signaling and/or by increasing interstitial pressure in the tumor resulting in decreased drug delivery1,6. Therefore, the addition of ECM and stromal cells into preclinical models will help recapitulate aspects of the tumor that cannot be modeled well in 2D culture.
Herein a method to establish breast cancer surrogates that incorporate a recapitulative microenvironment, including ECM constituents and stromal cells, in a 3D volume is described. In breast carcinoma, the stromal cell population is predominately comprised of cancer associated fibroblasts (CAF) and the stromal ECM is largely composed of collagen type I with a smaller proportion of matrix components that are found in the basement membrane, including laminin and collagen type IV1,4,11-13. Therefore, these components of the breast carcinoma microenvironment (i.e., CAF, collagen I, and basement membrane) have been incorporated into the surrogates. This method can be used to generate solid, un-perfused 3D surrogates (Figure 1A) or can be adapted to include perfusion of medium through the surrogate via a bioreactor system (Figure 1B). Both approaches are described here. This method could also be modified to include other stromal elements, such as tumor-associated macrophages, or to model other solid tumors by adjusting the cellular and ECM components, as appropriate.
For the breast carcinoma surrogate described here, we have utilized the MDA-MB-231 (231) breast cancer cell line, CAF previously isolated from human breast carcinoma14, and an ECM composed of 90% collagen I (6 mg/ml) and 10% growth factor reduced basement membrane material (BM). The surrogate is either grown in an 8-well chamber slide (solid surrogate) or a bioreactor system is utilized to provide continuous nutrient perfusion (perfused surrogate). Any perfusion bioreactor system that can accommodate a volume of ECM containing cells can be used15. As an example, we describe the preparation of the tissue surrogates in our bioreactor system. This system was developed in-house and is not commercially available. Because our focus here is on the preparation and analysis of the 3D tissue surrogates, we have not gone into extensive detail regarding the specifics of manufacture and assembly of our bioreactor system. However, a detailed description of this system and its development has been published16. In this bioreactor system, a polydimethylsiloxane (PDMS) flow channel is used to house the surrogate, which is supported by a PDMS foam (formed using methods similar to those described by Calcagnile et al.17). This volume is penetrated by 4 microchannels (each 400 µm in diameter) which are continuously perfused by medium via a microphysiologic pump to supply oxygen and nutrients to the surrogate.
Appropriate analysis of the surrogates is crucial to gain pertinent information regarding cellular function in response to treatment or other manipulations. Surrogates can be analyzed by various methods including direct imaging of intact surrogates using confocal microscopy or other means of non-invasive imaging, indirect cellular analysis by assaying the conditioned media, or perfusate, for secreted products, or analysis on histologic sections after fixation and processing to paraffin. One such parameter that can be evaluated on histologic sections is cell density. We present one approach to measure cell density (i.e., the number of nucleated cells per section area) using semi-automated image processing techniques applied to photomicrographs of surrogate histologic sections stained with hematoxylin and eosin (H&E). The cell density can be used as an indicator of the relative change in cell number over time or that results from varying growth conditions and treatments.
Figure 1. 3D volume and bioreactor system. A) Schematic of the process to generate solid 3D surrogates. Top: cartoon of solid 3D volume containing ECM (pink), epithelial carcinoma cells (yellow), and CAF (orange); Bottom: top view of 8-well chamber slide containing surrogates. B) Schematic of the process to generate perfused 3D surrogates. Top: cartoon of 3D volume with channels to allow for medium perfusion and containing ECM (pink), epithelial carcinoma cells (yellow), and CAF (orange); Middle: image of PDMS flow channel containing PDMS foam (black arrow) to be injected with cell+ECM and penetrated by polymer-coated stainless steel wires (pink arrow) measuring 400 µm in diameter; Bottom: image of the PDMS flow channel containing a surrogate and connected to the bioreactor system to allow for continuous medium perfusion (peristaltic pump and media reservoir not shown). C) Images of processing steps for both solid and perfused surrogates after culture. Left: image of the cryomold containing specimen processing gel and surrogate; Middle: image of a paraffin block containing a fixed and processed surrogate; Right: image of a glass slide with a H&E-stained histologic section of a surrogate. Please click here to view a larger version of this figure.
1. Cell Culture
2. Preparation of Cells in ECM (6 mg/ml Bovine Collagen Type I + 10% BM)
Note: An ECM composed of 90% collagen I + 10% BM was chosen to model invasive breast carcinoma because the tumor stroma in this malignancy is composed primarily of collagen I with components of the BM, such as laminin, collagen IV, and entactin, comprising a smaller portion of the ECM12,13,18,19.
Preparation of cells in ECM (6 mg/ml Bovine Collagen Type I + 10% BM) |
|
178.8 µl | Cell culture grade water containing the desired number of 231 cells (determined above) |
606 µl | Collagen I (10 mg/ml bovine), add drop by drop |
100 µl | Basement membrane, thawed |
100 µl | 10x DMEM (containing phenol red) with the desired number of CAF (determined above) |
15.2 µl | 7.5% (v/v) Sodium Bicarbonate, add drop by drop |
Table 1. Preparation of cells in ECM.
3. Surrogate Preparation
4. Surrogate Fixation and Processing (Figure 1C)
5. Sectioning and H&E Staining (Figure 1C, Right Panel)
H&E Staining | ||
Station | Solution | Time |
1 | Xylene | 5 min |
2 | Xylene | 5 min |
3 | Xylene | 5 min |
4 | 100% Ethanol | 5 min |
5 | 100% Ethanol | 5 min |
6 | 95% Ethanol | 5 min |
7 | 95% Ethanol | 5 min |
8 | Tap Water | 5 min |
9 | De-ionized Water | 5 min |
10 | Hematoxylin 7211 | 5 min |
11 | Tap Water | 5 min |
12 | Clarifier* | 10 dips |
*Richard Allan #7401 or 70% Ethanol + 0.5% HCl | ||
13 | Tap Water | 5 min |
14 | Bluing Reagent | 30 sec |
15 | Tap Water | 5 min |
16 | 95% Ethanol | 10 dips |
17 | Eosin-Y | 1 min |
18 | 95% Ethanol | 10 dips |
19 | 95% Ethanol | 10 dips |
20 | 100% Ethanol | 10 dips |
21 | 100% Ethanol | 10 dips |
22 | 100% Ethanol | 5 min |
23 | Xylene | 10 dips |
24 | Xylene | 5 min |
Table 2. H&E Staining.
6. Measuring Cell Density
Figure 2. ImageJ analysis. Screenshot of ImageJ processing. Please click here to view a larger version of this figure.
Figure 3. CellProfiler example pipeline. Screenshot of the pipeline designed to measure the number of nucleated cells in CellProfiler. Please click here to view a larger version of this figure.
Figure 4. CellProfiler pipeline: changing image to grayscale. Screenshot of "ColortoGray "module. Please click here to view a larger version of this figure.
Figure 5. CellProfiler pipeline: inverting image. Screenshot of "ImageMath" module. Please click here to view a larger version of this figure.
Figure 6. CellProfiler pipeline: identifying nuclei. Screenshot of "IdentifyPrimaryObjects" module. Please click here to view a larger version of this figure.
Figure 7. CellProfiler pipeline: identifying cells. Screenshot of "IdentifySecondaryObjects" module. Please click here to view a larger version of this figure.
Figure 8. CellProfiler pipeline: measuring objects. Screenshot of "MeasureObjectSizeShape" module. Please click here to view a larger version of this figure.
Figure 9. CellProfiler pipeline: filtering objects. Screenshot of "FilterObjects" module. Please click here to view a larger version of this figure.
Figure 10. CellProfiler pipeline: exporting data. Screenshot of "ExportToSpreadsheet" module. Please click here to view a larger version of this figure.
Figure 11. CellProfiler output following filtering. Screenshot of output screen in cell profiler following object filtering. Please click here to view a larger version of this figure.
Both solid and perfused 3D breast cancer surrogates were prepared as described above and grown for 7 days. Subsequently, surrogates were fixed, processed to paraffin, sectioned, and stained with hematoxylin and eosin, as described above. The number of nucleated cells per area (both 231 cells and CAF) of each surrogate was measured. As can be seen in Figure 12, representative photomicrographs of the H&E-stained sections demonstrate a higher concentration of cells present in the perfused surrogates (Figure 12A) compared to the solid surrogates (Figure 12B), even though the density of cells initially incorporated into the surrogates was the same in both solid and perfused conditions. This visual representation of cell growth is supported by a higher mean number of cells per area (density) in the perfused surrogates (n=3) compared to the solid surrogates (n=3), as determined by the CellProfiler analysis (Figure 12C). To obtain the data in Figure 12C, a complete histologic cross-section of each surrogate was imaged, requiring multiple images for each surrogate, and the images were analyzed as described above. Then, the total number of nuclei for each surrogate was divided by the total area of the surrogate (expressed as the number of nuclei/1 x 106 pixels2), providing a cell density for each surrogate. To validate the use of CellProfiler for nucleated cell quantification, the results using the CellProfiler program were compared with those obtained by simply manually counting the number of cells present per area in each of the 6 surrogates (Table 3). The cell density of each surrogate was calculated as described above. No significant difference was found in the cell densities obtained by manual counting and CellProfiler analysis for either perfused surrogates (p=0.855) or solid surrogates (p=0.553). To support that the difference in cell density between the solid and perfused surrogates correlates with a difference in cell growth, cell proliferation of each surrogate was evaluated via immunohistochemical analysis of Ki-67 labeling (Figure 12D)27. The same trend was found, with a significantly higher percentage of proliferating cells in the perfused surrogates (n=3) compared to the solid surrogates (n=3), indicating a higher growth rate in the perfused surrogates and correlating with the increased cell density of these surrogates. While the cell type (i.e., breast cancer epithelial or CAF) was not evaluated in these measurements, immunostaining for cell type-specific markers could be used to better understand the growth or distribution of a specific cell population. Protocols detailing this process have been previously published 25,28.
Figure 12. Representative Results. A) Representative image of a H&E-stained section from a perfused surrogate grown for 7 days (400X original magnification). B) Representative image of a H&E-stained section from a solid surrogate grown for 7 days with medium changed every 2 days (400X original magnification). C) Comparison of the mean number of nucleated cells per area, or cell density, following 7 days growth of perfused (no change of media) and solid surrogates (media changed every 2 days). D) Comparison of Ki-67 labeling index (i.e., the percentage of cells in a population that express Ki-67), a measure of cell proliferation, following 7 days growth of perfused and solid surrogates. Data represent the mean ± SEM, n=3 per condition, ** indicates p≤0.01 and *** indicates p≤0.001 (Student's t-test). Please click here to view a larger version of this figure.
Experiment | CellProfiler: nucleated cells per area | Manual: nucleated cells per area | Average CellProfiler | Average Manual | P value (Unpaired t Test) |
Perfused Surrogate 1 | 88.178 | 75.532 | 97.225 | 99.901 | 0.855 |
Perfused Surrogate 2 | 107.528 | 117.812 | |||
Perfused Surrogate 3 | 95.967 | 106.359 | |||
Solid Surrogate 1 | 19.797 | 17.480 | 16.491 | 12.991 | 0.553 |
Solid Surrogate 2 | 8.612 | 5.650 | |||
Solid Surrogate 3 | 21.065 | 15.844 |
Table 3. Cell densities obtained by manual or CellProfiler analysis for 3 solid and 3 perfused surrogates.
Herein, a method of 3D culture has been described that incorporates components of the tissue microenvironment, including the extracellular matrix (ECM) and human stromal fibroblasts, in a volume that more closely models human breast cancer to allow for the development of a recapitulative 3D morphology. The 3D culture method described is more representative of human disease than traditional 2D cell culture in that multiple cell types are incorporated into a 3D volume of ECM. It has been noted that these parameters (i.e., multiple cell types, ECM, and 3D volume) provide a more appropriated context to study biological processes because tissue architecture, signals from the microenvironment, and dimensionality are being taken into account 1,8. Other methods of 3D culture have been previously described including, the culture of cells on top of a 3D matrix29, the generation of spheroids30, hanging drop cutlures31, and the use of microfluidic devices32. While each of these systems has unique advantages, overall they fail to include either matrix components, stromal cell populations, or a representative dimensionality. The method of 3D culture described here includes each of these parameters and can be established using a bioreactor system to allow for longer growth periods that are difficult to achieve using solid 3D culture. In addition, perfusion of surrogates provides a greater degree of growth compared with solid surrogates.
Another feature of this approach for in vitro culture is the flexibility in the types of analyses that can be performed using the surrogate "tissues". A variety of endpoints can be monitored indirectly during culture by evaluating the conditioned media, or perfusate, for soluble products (e.g., LDH as an indicator of cell death, specific secreted proteins or metabolites as an indicator of appropriate phenotypic function) or by utilizing non-invasive imaging of fluorescently or luminescently labeled cells to evaluate growth and surrogate architecture overtime. Following growth, the ECM can be digested using an ECM protease and cells can be isolated and used for additional analysis or further experiments, such as flow cytometric quantification33. Lysates of the cells removed from the ECM could also be evaluated genomically, at the mRNA transcript level, or for protein expression. Alternatively, endpoints can be measured directly from the fixed and processed surrogate "tissues" after the experiment has concluded. These include a variety of morphological parameters (e.g., nuclear/cell morphology and the degree of cell aggregation) on H&E-stained histologic sections and other molecular analyses performed on histologic sections using immunohistochemistry (e.g., expression of Ki-67 as an indicator of cell proliferation) or in situ hybridization (e.g., RNA expression of specific gene products). It is also possible to perform additional molecular analyses, such as real-time, quantitative PCR or next generation sequencing, on these formalin-fixed, paraffin-embedded surrogates; however, due to molecular cross-linking induced by fixation and processing, snap-frozen surrogates are preferred for these types of analyses. Such molecular and morphologic evaluations can provide valuable information regarding cell growth, death, or response to pharmacological treatment throughout growth as well as following culture.
The main advantage of using the CellProfiler program for semi-automated quantification of the density of nucleated cells present in histologic sections of surrogates is the time savings that it provides. Although the total amount of time required per surrogate is variable and depends largely on the size of the surrogates and the number of images acquired, it is estimated that arriving at a cell density from the surrogate images requires 4-5 hours of hands-on time per surrogate when manually counting versus half an hour per surrogate when using the CellProfiler program. The majority of the hands-on-time using CellProfiler analysis is attributed to the measurement of the surrogate area in ImageJ. The CellProfiler program does require time to automatically process the images, but does not require hands-on time from the researcher other than importing the images.
There are limitations of the methods and the 3D surrogate cultures described here. As shown in Figure 12, the use of the perfusion bioreactor system provided a greater degree of growth than solid cultures, in which the accumulation of cells and the rate of proliferation is considerably slower. Although the perfusion bioreactor system used here provided a growth advantage, perfusion systems can also have their own limitations. For example, PDMS is often used in the manufacture of bioreactors due to its ease of use, inert properties, and reproducibility. However, this material is known to absorb hydrophobic molecules from cell culture media and may take up lipophilic drugs34-36. While other materials can be used instead of PDMS if these limitations are of concern, other materials available also come with their own drawbacks that must be considered. Because CellProfiler analysis is dependent on the parameters input for the diameter and circularity of the nuclei, optimization of these inputs for each surrogate/cell type may be necessary. It is also of note that the cell density measurement presented here is not a direct measurement of cell viability. It has previously been noted that a direct measurement of cell viability is challenging in 3D culture systems37. However, nuclear degradation is a part of both apoptosis and necrosis, the two predominate forms of cell death. In apoptosis, the nucleus fragments, a process called karyorrhexis, following chromatin condensation, or pyknosis38,39. This differs from necrosis, where the dying cell swells causing the cell membrane to rupture and release the contents of its cytoplasm. Histologically, necrosis is characterized by karyolysis (i.e., dissolution of a nucleus), followed by pyknosis and karyorrhexis40. Nuclei that have undergone these changes can be seen on H&E-stained histologic sections but will not be recognized as intact nuclei by the CellProfiler program. These nuclear changes occur relatively late in the process of cell death however. Therefore, quantifying the number of nucleated cells per area may include cells that are in the earlier stages of cell death, but will nonetheless provide useful information regarding cell viability at the time of surrogate fixation. Other methods that could be used to indicate viability or growth include analyses of perfusates (e.g., Alamar Blue or LDH assays) or evaluation of proliferation in the surrogate "tissues" (e.g., via Ki-67 labeling by immunohistochemistry).
Critical steps in the protocol for surrogate setup include ECM polymerization, where temperature, pH, and time all must be monitored to ensure complete polymerization prior to bathing the surrogate in medium. In addition, keeping the cell+ECM mixture on ice prior to plating in an 8-well chamber slide or injection into a bioreactor system is crucial to preventing premature polymerization. Also to prevent polymerization during surrogate setup, the sodium bicarbonate used to increase the pH to 7 should be added to the mixture last. Finally, adequate incubation time at 37 °C must be given to ensure complete ECM polymerization; 45 minutes has proven to be sufficient. Another important factor in surrogate preparation is the avoidance of bubble formation. In both the solid and perfused systems, it is best to avoid the formation of bubbles in the cell+ECM mixture as to not create a block to the flow of nutrients during culture. It is also important to avoid bubbles during medium introduction in the perfusion system where a bubble may function as an air embolism, blocking medium flow.
Proper fixation is important for downstream analysis; therefore, attention to standardized and consistent fixation (including the type of fixation and the amount of time in fixative) and processing is necessary to obtain replicative results. The duration and type of fixation is less of an issue for the H&E staining described here, but these parameters may affect other types of molecular analysis of the surrogates. This is because formalin fixation, in particular, causes the cross-linking of molecules, including protein, DNA, and RNA in a somewhat time-dependent and molecule-specific manner41,42. This is of particular importance in immunohistochemical analysis, when antibody recognition may be hindered by cross-linking.
The authors have nothing to disclose.
The University of Alabama at Birmingham Center for Metabolic Bone Disease performed the histologic processing and sectioning of surrogates. Southern Research (Birmingham, AL) provided support for the manufacture of the bioreactor system. Funding was provided by the United States Department of Defense Breast Cancer Research Program (BC121367).
Dulbecco's Modified Eagel Medium 1x (DMEM) | Corning CellGro | 10-014-CV | |
Fetal Bovine Serum (FBS) | Atlanta Biologicals | S11150 | |
0.25% Trypsin + 2.21 mM EDTA 1x | Corning | 25-053-CI | |
Tissue Culture plates, 100mm | CellTreat Scientific Products | 229620 | Sterile |
Tissue Culture plates, 35mm | CellTreat Scientific Products | 229638 | For PDMS foam formation |
9" Glass pipette | Fisher | 13-678-20D | Sterile |
10 ml pipette | CellTreat Scientific Products | 229210B | Sterile |
1000 µl piptette tips | FisherBrand | 02-717-166 | Sterile Filtered |
200 µl pipette tips | FisherBrand | 02-717-141 | Sterile Filtered |
10 µl pipette tips | FisherBrand | 02-717-158 | Sterile Filtered |
15 ml conical tubes | CellTreat Scientific Products | 229410 | Sterile |
50 ml conical tubes | CellTreat Scientific Products | 229422 | Sterile |
1.5 ml microcentrifuge tubes | FisherBrand | 05-408-129 | Sterile |
Trypan blue | Corning Cellgro | 25-900-CI | Sterile |
Sylgard 184 | Electron Microscopy Sciences | 24236-10 | PDMS elastomer and curing agent. Used for our in-house bioreactor. |
PDMS Foam | Made in-house for use in our in-house bioreactor. | ||
High Concentration Bovine Collagen Type I | Advanced Biomatrix | 5133-A | FibriCol ~10 mg/mL |
Growth Factor Reduced Matrigel (Basement Membrane) | Corning | 354230 | Basement membrane material |
Sodium Bicarbonate | Sigma | S8761 | |
Molecular Biology Grade Water | Fisher | BP2819-1 | |
DMEM 10x | Sigma-Aldrich | D2429 | |
Nunc Lab-Tek Chamber Slide System | Thermo Scientific | 177402 | 8-well |
Bioreactor | Made in-house. | ||
Spring-Back 304 Stainless Steel—Coated with PTFE polymer | McMaster-Carr | 1749T19 | Stainless steel wires to generate microchannels in our in-house bioreactor system. 0.016" Diameter |
BioPharm Plus platinum-cured silicone pump tubing, L/S 14 | Masterflex | EW-96440-14 | For use in our in-house bioreactor system. Tubing ID: 1.6 mm, Hose barb size: 1/16 in. |
2-Stop Tubing Sets, non-flared PVC, 1.52 mm ID | Cole-Parmer | EW-74906-36 | For use in our in-house bioreactor system (with microperistalitic pump). |
Six Channel precision micro peristaltic pump | Cole-Parmer | EW-74906-04 | For use with our in-house bioreactor system |
Labtainer BPC Bag – 2 Ports, Luer Lock 50mL |
Thermo Scientific | SH3065711 | Example Media Reservoir |
Tuberculin Syringes | BD Medical | 309625 | 26 gauge 3/8 in. needle; Sterile |
Dissecting Tissue Forceps | FisherBrand | 13-812-36 | 5.5 inch |
Mini Tube Rotator | Boekel Scientific | 260750 | Equipment option for surrogate rotation. Used with carousel for 50 ml tubes (model number 260753) |
50 ml tube carousel | Boekel Scientific | 260753 | Used with mini tube rotator |
Bambino Hybridization Oven | Boekel Scientific | 230301 | Equipment option for surrogate rotation |
HistoGel Specimen Processing Gel | Thermo Scientific | HG-4000-012 | Specimen Processing Gel described in Step 5.2 |
Cryomold | Andwin Scientific | 4566 | 15 mm x 15 mm x 5 mm |
Tissue Marking Dye | Cancer Diagnostics, inc. | 03000P | Can be used to mark surrogates, allowing multiple samples to be included in one tissue cassette |
Hinged tissue cassettes | FisherBrand | 22-272-416 | |
Formalin | Fisher | 23-245-685 | |
GoldSeal Plain Glass Slides | Thermo Scientific | 3048-002 | |
Xylene | Fisher | X3P-1GAL | |
Ethanol, 200 proof (100%), USP | Decon Laboratories, Inc. | 2805M | |
Hematoxylin | Thermo Scientific Richard-Allan Scientific | 7211 | |
Clarifier | Thermo Scientific Richard-Allan Scientific | 7401 | |
Bluing Solution | Thermo Scientific Richard-Allan Scientific | 7301 | |
Eosin Y | Thermo Scientific Richard-Allan Scientific | 7111 | |
Cytoseal XYL mounting media | Thermo Scientific Richard-Allan Scientific | 83124 | |
Coverslips | Fisher Scientific | 12-548-5G |