Using three-dimensional organotypic cultures to visualize morphology and functional markers of salivary glands may provide novel insights into the mechanisms of tissue damage following radiation. Described here is a protocol to section, culture, irradiate, stain, and image 50–90 μm thick salivary gland sections prior to and following exposure to ionizing radiation.
Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.
Proper salivary gland function is essential to oral health and is altered following head and neck cancer treatment with radiotherapy1. In 2017, nearly 50,000 new cases of head and neck cancer were reported in the United States2. Due to the tissue-damaging and frequently irreversible effects of radiation therapy on surrounding normal tissues such as salivary glands, patients are often left with severe side effects and diminished quality of life2,3,4. Common complications caused by radiation damage manifests in symptoms such as xerostomia (the subjective feeling of dry mouth), dental caries, impaired ability to chew and swallow, speech impairments, and compromised oral microbiomes2,3,4. These symptoms collectively can lead to malnutrition and impaired survival in affected individuals5. While salivary gland dysfunction in this population has been well-documented, the underlying mechanisms of damage to acinar cells have been disputed, and there is little integration among different animal models6,7.
The current methods of studying salivary gland function and radiation-induced damage include the use of in vivo models, immortalized cell lines, two-dimensional (2-D) primary cell cultures, and three-dimensional (3-D) salisphere cultures8,9,10,11,12. Traditionally, cell culture models from immortalized cell lines and 2-D cultures involve single layered cells cultured on flat surfaces and are valuable for fast, easy, and cost-effective experimentation. However, artificial cell culture conditions can alter the differentiation status and physiological responses of cells exposed to various conditions, and the results often fail to translate to whole organism models14,15. In addition, immortalized cell cultures require modulation of p53 activity, which is critical for the salivary gland response to DNA damage16,17.
3-D salisphere cultures are enriched for stem and progenitor cells at early time points in culture and have been useful for understanding the radiosensitivity of this subset of salivary gland cells9,18. A critical limitation of all these culture models is they are ineffective in visualizing the 3-D structure of the salivary gland, including the extracellular matrix (ECM) and cell-cell interactions over various layers, which are crucial in modulating salivary secretion15. The need for a method that encompasses the behavior of the tissue as a whole but can also be manipulated under laboratory conditions to study the effects of treatment is necessary to further discover the underlying mechanisms of radiation-induced salivary gland dysfunction.
Live tissue sectioning and culture has been documented previously19,20 and is often used to study brain-tissue interactions21. In previous studies, parotid (PAR) salivary gland tissue from mice was sectioned at approximately 50 µm and cultured for up to 48 h, and analysis of viability, cell death, and function was performed thereafter19. Su et al. (2016) expanded on this methodology by culturing human submandibular glands (SMGs) sectioned at 35 µm or 50 µm for 14 days20. The proposed method is an advancement in that it includes both parotid and submandibular salivary glands sectioned at 50 µm and 90 µm and evaluation of the cultures for 30 days. The ability to cut a range of tissue thickness is important in evaluating cell-cell and cell-ECM interactions that are relevant for cellular processes including apical-basolateral polarity and innervation for secretion. Furthermore, the salivary gland slices were irradiated while in culture to determine the feasibility of this culture model to study radiation-induced salivary gland damage.
The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. To determine the maximum time sections that are viable post-dissection, trypan blue staining, live cell staining, and immunohistochemical staining for cell death were performed. Confocal microscopy and immunofluorescent staining were utilized to evaluate specific cell populations, morphological structures, and levels of proliferation. Tissue section cultures were also exposed to ionizing radiation to determine the effects of radiation on various markers in this 3-D model. Induction of cell death, cytoskeletal disruption, loss of differentiation markers, and compensatory proliferation in irradiated ex vivo cultures were compared to previous studies of in vivo models. This methodology provides a means to investigate the role of cell-cell interactions following radiation damage and provides an experimental model to efficiently evaluate the efficacy of therapeutic interventions (gene manipulations or pharmacological agents) that may be less suitable for in vivo models.
1. Preparation of vibratome
2. Preparation of tissue sample in agarose block and sectioning using the vibratome
3. Culturing sections
4. Irradiation of salivary gland sections
5. Viability staining
6. Antibody staining vibratome sections
NOTE: The following provides a general antibody staining protocol specific for Ki-67; however, this protocol can be used with any antibody. All washes are conducted at RT unless otherwise noted.
7. Imaging vibratome sections
Primary 2-D cultures are grown in fetal bovine serum (FBS) supplemented media while primary 3-D salisphere culture are typically cultured in serum-free conditions10,11. In addition, the two previous studies utilizing vibratome cultures from salivary glands cultured their sections in 0% or 10% FBS supplemented media19,20. Mouse submandibular slices were sectioned at a thickness of 50 µm using a vibratome and optimal culture conditions were determined using a series of FBS concentrations (0%, 2.5%, 5.0%, and 10%). To determine survival characteristics, bright-field microscope images were taken at culture days 1, 4, 7, 14 ,and 30 post-sectioning (Figure 1). Additionally, gland sections were stained with 0.4% trypan blue dye and imaged with a bright-field microscope at 40x at the indicated time points (Figure 2A). Non-viable cells were stained blue and viable cells remained clear.
Similarly, to determine the survival characteristics of thicker slices sectioned at 90 µm, bright-field microscope images with and without trypan blue staining were taken at day 30 in cultures supplemented with 2.5% FBS (Figure 2B). Due to the slice thickness, 90 µm sections stained with trypan blue dye were imaged whole then cut in half in order to evaluate the center of the slice (Figure 2B). As a confirmation, a live-cell dye was utilized to evaluate cell survival in different FBS culturing conditions (Figure 2C). In bright-field images, high levels of translucent, surviving cells in tissues sectioned at both 50 µm and 90 µm were observed. Interestingly, sections became darker and overall tissue area condenses over time in culture, yet a significant portion of the section appears to survive the 30-day culture period (Figure 1, Figure 2). This condensation was most evident in the 0% and 10% FBS culturing conditions. Trypan blue positive cells were observed on the perimeter of all sections regardless of culturing conditions, and there was an increase in trypan blue positive cell area in 0% FBS culture conditions when compared to the higher FBS culturing conditions (Figure 2A,2B).
Using the live cell stain, the sections cultured in 0% showed the lowest amount of staining, and the addition of FBS to the culture media improved the amount of live cells. Taken together, sections cultured in 0% FBS showed visible tissue condensation, elevated trypan blue stained areas, and the lowest levels of live cell staining. The addition of 2.5% FBS to the cultures improved the amount of translucent tissue, decreased the trypan blue positive area, and increased the levels of live cell staining. Additional increases in FBS concentration did not appear to improve survivability of the tissue; therefore, 2.5% FBS in the vibratome media was the optimal FBS concentration and utilized as the culture condition for all subsequent experiments.
To determine the viability of the submandibular ex vivo tissue slices post-dissection, proliferative and apoptotic markers were evaluated at days 1, 3, 7, 14, and 30 in culture. The proliferative activity was assessed by Ki67 immunostaining in a subset of culture slices (Figure 3A). Ki67 positive cells were observed at all time points evaluated and continued to be present at day 30 in culture, with minimal differences between time points. Similarly, the degree of apoptosis was evaluated by cleaved caspase-3 immunostaining in a separate subset of sections (Figure 3B). A low level of cleaved caspase-3 positive cells was observed at all time points up to day 14 in culture, while day 30 conditions appeared to have a small increase in the number of cleaved caspase-3 positive cells in some areas. Evaluation of the tissue edges did not reveal higher levels of cleaved caspase-3, which does not recapitulate the trypan blue staining (Figure 2). Overall, these results suggest that cues for proliferation and viability remained present in the vibratome cultures during the 30-day evaluation period.
Thick section vibratome cultures allow the opportunity to evaluate interactions between cellular constituents of a particular tissue at a depth that includes more than one epithelial cell thickness in each direction. In addition, it is important to be able to culture both major salivary glands, as they differ in the composition of salivary proteins produced, histological architecture, radiosensitivity, and other critical features. To determine different cellular populations in submandibular gland cultures, submandibular sections were stained with E-cadherin (E-cad) to detect epithelial cells, smooth muscle actin (SMA) to detect myoepithelial cells, and actin filaments (phalloidin) to detect cytoskeletal structures during the 30 days in culture (Figure 4A).
E-cadherin staining was observed on the membranes of a majority of cells and detected throughout the 30-day culture period. SMA+ cells were detected throughout the culture period, with similar levels at each time point. Cytoskeletal organization of actin filaments also appeared to be maintained at each time point evaluated. In contrast, there were cellular markers that were not consistently maintained during the entire evaluation period and these included CD31 (vasculature), TUBB3 (neurons), and Aquaporin-5 (Aqp-5, acinar marker) (Figure 4B). Vascular structures through the confocal stacks were clearly observed at days 1 and 3 post-culture; however, these structures appeared fragmented at day 7. Similarly, neuronal processes were intact during the first day of culture, appeared diminished at day 3, and were subsequently lost at day 7 in culture. Aqp5+ cells were observed at days 1, 3, 7, and 14 in culture; albeit, at day 14, the overall staining level appeared to be reduced, with a more granular resemblance in the remaining positive cells. These data suggest that submandibular cultures contain the diversity of tissue constituents with maintenance of the vascular and neuronal cell types for shorter culture periods, and epithelial and myoepithelial cell types for longer culture periods.
While vibratome-sectioned cultures have been previously reported for the parotid salivary gland, the cultures were maintained for 48 h, limiting the timeframe during which they could be studied. In order to determine whether parotid glands can be maintained for longer, murine parotid glands were sectioned and cultured for 1, 3, 7, or 14 days. Fewer sections were obtained from the parotid gland due to its smaller size in mice; therefore, a 30-day culture period was not attempted. Slices were evaluated for proliferation by immunofluorescent staining using antibody against Ki67. Similar to the submandibular cultures, Ki67 positive cells were observed at all time points, indicating that the cells are maintaining a degree of proliferation in culture (Figure 5A).
In addition, the maintenance of functional acinar markers (α-amylase and Aqp5), an epithelial marker (E-cad), and vascular (CD31) and neuronal (TUBB3) cell populations were evaluated. Amylase is one of the most abundant proteins produced by differentiated parotid epithelial cells and frequently lost during the 2-D culturing of primary parotid cells. In the vibratome cultures, amylase was observed in the acinar cells and excluded from the ductal cells throughout the 14-day culture period (Figure 5B). Similar to the submandibular cultures, Aqp5+ cells were present in the parotid cultures at each time point, with the day 14 cultures exhibiting reduced levels compared to earlier time points (Figure 5C). E-cadherin levels were also maintained on the membranes of a majority of cells during the 14-day culture period (Figure 5D). Neuronal structures appeared to be maintained during the 7 day culture period and were not assessed at later time points (Figure 5E). In contrast, vascular structures appeared intact during the first day in culture, and only smaller structures were present at days 3 and 7 in culture (Figure 5F). These data suggest that parotid cultures maintain their proliferative capabilities and most functional capabilities for 7-14 days in culture and potentially exhibit a more intact tissue structure for a longer time frame.
The functional utility of the vibratome culture model was addressed by treating parotid or submandibular cultures with a single dose of radiation (Figure 6, Figure 7, Figure 8). Previous work in irradiated mouse models has focused on the parotid gland and demonstrated the induction of apoptosis that peaks at 24 h, induction of compensatory proliferation starting at day 5, disruption of actin filaments starting at day 5, and loss of differentiation markers (e.g., amylase) by day 1423,24,26. Irradiation of parotid vibratome cultures led to increases in apoptosis at day 1, increases in proliferation at day 7, disruption of actin filaments at day 7, and reductions in amylase at day 7 (Figure 6). Irradiation of submandibular vibratome cultures led to increases in apoptosis at days 1 and 3, increases in proliferation at day 7, and disruption of actin filaments at day 7 (Figure 7). E-cadherin levels appeared relatively intact in both parotid and submandibular cultures, which was similar to in vivo observations26. The functional acinar cell markers Aqp-5 in submandibular gland sections and amylase in parotid gland sections decreased at day 14, compared to corresponding untreated time points (Figure 8). These data suggest that radiation-induced tissue changes that were observed in vivo were also observed in irradiated vibratome cultures.
Figure 1: Bright-field microscope images of 50 µm submandibular sections. Submandibular glands from female FVB mice (4-8 weeks old) were dissected, sectioned to 50 μm thickness, and cultured on organotypic cell culture inserts for 1, 4, 7, 14, and 30 days post-dissection at 0%, 2.5%, 5.0%, and 10% fetal bovine serum (FBS) in media to determine culture characteristics and optimize culture conditions. Scale bars = 200 μm. Please click here to view a larger version of this figure.
Figure 2: Viability staining of submandibular sections. (A) Bright-field microscope images of 50 µm submandibular dissected from 4- to 8-week old female FBV mice and stained with trypan blue (0.4%) at 1, 3, 7, 14, and 30 days in culture with media containing 2.5% fetal bovine serum (FBS). (B) Bright-field microscope images of 90 µm submandibular sections (left panel), stained with trypan blue (middle panel), cut in half and stained with trypan blue (right panel), then cultured to 30 days post-dissection in media containing 2.5% FBS. (C) Fluorescent images of 50 µm submandibular sections cultured in various FBS concentrations (0%, 2.5%, 5%, 10%) stained with calcein AM to indicate live cells (green) at culture day 7. Scale bars = 200 μm. Please click here to view a larger version of this figure.
Figure 3: Evaluation of proliferative and apoptotic markers in submandibular organotypic tissue slices. Submandibular glands from female FVB mice (4-8 weeks old) were dissected, sliced to 50 µm thickness, and cultured in media supplemented with 2.5% FBS on organotypic cell culture inserts for 1, 3, 7, 14, and 30 days post-dissection. At the indicated time points, slices were fixed and stained for proliferative (Ki67) and apoptotic (cleaved caspase-3) markers. (A) Immunofluorescent staining of Ki67-positive cells (green) and the nucleus (blue). (B) Immunofluorescent staining of cleaved caspase-3-postive cells (red) and the nucleus (blue) from two viewpoints of a slice. The top row panel displays cleaved caspase-3-positive cells on the edge of a slice, and the bottom row panel displays cleaved caspase-3-positive cells from the middle of a slice. Representative confocal images were selected from multiple z-stacks per time point. Scale bars = 30 µm. Please click here to view a larger version of this figure.
Figure 4: Presence of cellular structures in submandibular organotypic tissue slices. Submandibular glands from female FVB mice (4-8 weeks old) were dissected, sliced to 50 μm thickness, and cultured in media supplemented with 2.5% FBS on organotypic cell culture inserts for 1, 3, 7, 14, or 30 days post-dissection. At the indicated time points, slices were fixed and stained for their corresponding markers with the nucleus (blue). (A) Submandibular sections were evaluated at days 1, 7, 14, and 30 post-dissection for the levels of E-cadherin, smooth muscle actin (SMA), and F-actin (phalloidin). (B) Submandibular sections were evaluated at days 1, 3, and 7 for levels of CD31 (vasculature) and TUBB3 (neurons). Aquaporin-5 (Aqp5) was evaluated at days 1, 3, 7, and 14. Representative confocal images were selected from multiple z-stacks per time point. Scale bars = 30 μm. Please click here to view a larger version of this figure.
Figure 5: Evaluation of proliferative and functional markers in PAR organotypic tissue slices. Parotid glands from female FVB mice (4-8 weeks old) were dissected, sliced to 50 µm thickness, and cultured in media supplemented with 2.5% FBS on organotypic cell culture inserts for 1, 3, 7, or 14 days post-dissection. At the indicated time points, slices were fixed and stained for their corresponding markers with the nucleus (blue). (A) Immunofluorescent staining of Ki67-positive cells (green). (B) Immunofluorescent staining of amylase-positive cells (red). (C) Immunofluorescent staining of aquaporin-5 (Aqp5)-positive cells (green). (D) Immunofluorescent staining of E-cadherin (red) positive cells. (E) Immunofluorescent staining of neurons indicated by TUBB3 (magneta) positive cells. (F) Immunofluorescent staining of the vasculature indicated by CD31 (red) positive cells. Representative confocal images were selected from multiple z-stacks per time point. Scale bars = 30 μm; d = ductal cells. Please click here to view a larger version of this figure.
Figure 6: Cellular changes following irradiation of parotid organotypic tissue slices. Parotid glands from female FVB mice (4-8 weeks old) were dissected, sliced to 50 μm thickness, and cultured in supplemented with 2.5% FBS on organotypic cell culture inserts. On day 1 post-dissection, a subset of slices was exposed to 5 Gy radiation and maintained for 2, 4, or 8 days post-dissection (corresponds to days 1, 3, and 7 post-radiation). Immunofluorescent staining of untreated and irradiated parotid sections to determine levels of (A) Ki67 (green)-positive cells, (B) cleaved caspase 3 (red)-positive cells, (C) phalloidin (cyan)- positive cells, (D) amylase (red)-positive cells, and (E) E-cadherin (red) positive cells. All nuclear staining utilized DAPI (blue). Representative confocal images were selected from multiple z-stack per time point. Scale bars = 30 μm. Please click here to view a larger version of this figure.
Figure 7: Cellular changes following irradiation of submandibular organotypic tissue slices. Submandibular glands from female FVB mice (4-8 weeks old) were dissected, sliced to 50 μm thickness, and cultured in media supplemented with 2.5% FBS on organotypic cell culture inserts. On day 1 post-dissection, a subset of slices was irradiated with 5Gy and maintained for 2, 4, or 8 days post-dissection (corresponds to days 1, 3, and 7 post-radiation). Immunofluorescent staining of untreated and irradiated submandibular sections to determine levels of (A) Ki67 (green)-positive cells, (B) cleaved caspase 3 (red)-positive cells, (C) phalloidin (cyan)- positive cells, (D) aquaporin 5 (Aqp5) (green)-positive cells, and (E) E-cadherin (red) positive cells. All nuclear staining utilized DAPI (blue). Representative confocal images were selected from multiple z-stack per time point. Scale bars = 30 μm. Please click here to view a larger version of this figure.
Figure 8: Functional acinar markers in irradiated parotid and submandibular organotypic tissue slices. Submandibular and parotid glands from female FVB mice (4-8 weeks old) were dissected, sliced to 50 μm thickness, and cultured in media supplemented with 2.5% FBS on organotypic cell culture inserts. On day 1 post-dissection, a subset of slices was irradiated with 5Gy and maintained for 14 days post-irradiation. Immunofluorescent staining of untreated (UT) and irradiated (IR) sections were used to determine levels of (A) aquaporin-5 (Aqp5) (green)-positive cells and (B) amylase (red)-positive) cells. All nuclear staining utilized DAPI (blue). Representative confocal images were selected from multiple z-stack per time point. Scale bars = 30 μm. Please click here to view a larger version of this figure.
Salivary gland research has utilized a number of culture models, including immortalized 2-D cultures, primary 2-D cultures, 3-D salisphere cultures, and 3-D organ cultures from embryonic explants to ascertain questions on underlying biology and physiology. These culture models have yielded insightful information across a diverse array of research questions and will continue to be important tools in salivary research. The limitations of these culture models include modulation of p53 activity during immortalization, transient viability of primary cultures, loss of differentiation and secretory proteins in culture, and inability to evaluate cell-cell, cell-ECM and polarity interactions in adult tissues. The first 3-D organotypic slice culture (vibratome sectioned cultures) method for salivary glands was published in 200818; however, this technique has been largely underutilized in this field despite frequent use in other fields. The work cultured parotid sections for 48 h, which limits the ability to study these sections for chronic effects following radiation treatment or utilize transfection or transduction protocols for phenotypes following manipulation of specific genes. The method described here has been optimized to allow for longer time in culture, yield high resolution images through confocal microscopy, provide a method to study intra- and intercellular dynamics on a 3-D section, and evaluate radiation-induced changes during a culture period of at least 14 days.
While each step is required for implementation of the technique, some steps are crucial to successful sectioning and culture maintenance. These include cutting the salivary gland slices, maintaining the slices in culture, and staining and imaging the slices with confocal microscopy. The presented protocol poses some challenges that require patience and practice in order to obtain optimal slices for analysis. The following suggestions will assist in carrying out this protocol successfully. It is imperative to completely isolate the salivary gland from the surrounding connective tissue following dissection. Residual connective tissue causes the vibratome blade to drag the gland out from the agarose block and requires the tissue to be re-embedded in agarose. This can be a major limitation since multiple re-embedding can increase the chances of contamination and decrease the viability of the slices. This is especially crucial for culturing parotid glands, since the parotid glands are more lobular in structure and therefore more likely to have extraneous tissue.
The addition of 1% penicillin-streptomycin-amphotericin B (PSA) to all liquids including the buffer tray, the slice collection dish, and the vibratome media minimizes contamination of the slices post-dissection. Variation of agarose concentration was used to optimize successful cutting. Due to the density of the salivary glands, 1.9% agarose was too soft, and the glands were easily dislodged from the block. Vibratome sectioning in other fields have used 5.0% agarose; however, this caused jagged cuts and slices were suboptimal. After testing several agarose percentage conditions, 3% agarose was the most optimal to support the weight and firmness of the tissue. Notably, the agarose concentration utilized in Warner et al. and Su et al. was also 3%19,20.
Additionally, the angle, frequency of vibration, and advancing speed of the blade can be modified based on the tissue to be sectioned. For submandibular and parotid salivary glands, a 15° angle, speed of 0.075 mm/s, and frequency of 100 Hz were appropriate for sectioning. Due to the softness of the salivary glands, the optimal cutting conditions required the blade to advance slowly through the tissue at a high vibration. For immunofluorescent staining, the permeabilization, duration of incubations, and wash steps are essential for optimal stains. If positive staining only appears in the outer layers of the tissue slice, a more stringent permeabilization with proteinase K may be needed, while uneven staining or high background staining may require a less stringent staining with 0.2% Triton X-100. The incubation times were optimized for high signal and low background, which may need to be tailored to specific primary antibodies. Longer wash steps are essential in reducing high background and this may be tailored to the specific antibodies used.
One major application of this methodology is extended kinetic analysis following radiation exposure of salivary glands. Prior work has established both acute and chronic phase changes in irradiated salivary glands6,24,25,26, and the vibratome culture system can be a powerful tool to dissect out the critical molecular events at specific time points after treatment. For example, radiation-induced cellular changes in the salivary gland that have been reported in the literature, including reductions in amylase, apoptosis of acinar cells, compensatory proliferation of acinar cells, loss of polarity and disruptions in cytoskeletal structure. Notably, vibratome cultures irradiated ex vivo exhibit similar alterations in these markers.
In addition, the acute phase response in salivary glands pivots around p53 activity; however, it is unclear what role p53 plays in later time points due to the ~5-day viability of primary cultures. This system would allow ex vivo, controlled disruption of p53 activity at later time points and uncover a role in chronic damage or regenerative responses. Furthermore, the compensatory proliferation response is initiated 5 days after radiation treatment, and it is difficult to delineate the molecular regulators of this response in transient primary cultures. The most widely used application of this methodology will likely involve cell-cell, cell-ECM, and polarity interactions in adult tissues. Impactful studies have been conducted in 3-D organ cultures from embryonic glands to uncover the intricate interaction between developing salivary glands and the neuronal or vascular network27,28,29,30,31.
The method described here indicates that further optimization is needed for neuronal or vascular work in the submandibular cultures and possibly parotid cultures. Radiation damage also disrupts junctional regulators, induces collagen deposition, alters F-actin organization, and modulates secretory granules26,30,32. Salivary gland regeneration studies are handicapped by the absence of an adult model to evaluate these interactions. This organotypic culture method can provide a system to apply advanced molecular techniques and further study the regulation of these mediators in a 3-D context and efficiently discover new therapies.
The authors have nothing to disclose.
This work was supported in part by pilot funding provided by University of Arizona Office of Research and Discovery and National Institutes of Health (R01 DE023534) to Kirsten Limesand. The Cancer Biology Training Grant, T32CA009213, provided stipend support for Wen Yu Wong. The authors would like to thank M. Rice for his valuable technical contribution.
Vibratome VT1000S | Leica Biosystems | N/A | Vibratome for sectioning |
Double Edge Stainless Steel Razor Blades | Electron Microscopy Sciences | 72000 | |
Agarose | Fisher Scientific | BP165-25 | Low-melt |
Parafilm | Sigma-Aldrich | P6543 | |
Penicillin-Streptomycin-Amphotericin B | Lonza | 17-745H | PSA |
24-well plate | CellTreat | 229124 | |
Dulbecco’s Phosphate Buffered Saline (DPBS) | Gibco | 14190-144 | |
Loctite UltraGel Control Superglue | Loctite | N/A | Purchased at hardware store |
Natural Red Sable Round Paintbrush | Princeton Art & Brush Co | 7400R-2 | |
Gentamicin Sulfate | Fisher Scientific | ICN1676045 | |
Transferrin | Sigma-Aldrich | T-8158-100mg | |
L-glutatmine | Gibco | 25030-081 | |
Trace Elements | MP Biomedicals | ICN1676549 | |
Insulin | Fisher Scientific | 12585014 | |
Epidermal Growth Factor | Corning | 354001 | |
Hydrocortisone | Sigma-Aldrich | H0888 | |
Retinoic acid | Fisher Scientific | R2625-50MG | |
Fetal Bovine Serum | Gibco | A3160602 | |
DMEM/F12 Media | Corning | 150-90-CV | |
Millicell Cell Culture Insert | Millipore Sigma | PICM01250 | 12 mm, 0.4 um pore size for 24 well plate |
0.4% Trypan Blue | Sigma-Aldrich | T8154 | |
LIVE/DEAD Cell Imaging Kit (488/570) | Thermo-Fisher | R37601 | Only used LIVE dye component |
Anti-Ki-67 Antibody | Cell Signaling Technology | 9129S | |
Anti-E-cadherin Antibody | Cell Signaling Technology | 3195S | |
Anti-Cleaved Caspase-3 Antibody | Cell Signaling Technology | 9661L | |
Anti-SMA Antibody | Sigma-Aldrich | C6198 | |
Anti-amylase Antibody | Sigma-Aldrich | A8273 | |
Anti-CD31 Antibody | Abcam | ab28364 | |
Anti-TUBB3 Antibody | Cell Signaling Technology | 5568S | |
Alexa Fluor 594 Antibody Labeling Kit | Thermo-Fisher | A20185 | |
Alexa Fluor 594 Phalloidin | Thermo-Fisher | A12381 | |
Bovine Serum Albumin | Fisher Scientific | BP1600 | |
Triton X-100 | Sigma-Aldrich | 21568-2500 | |
Paraformaldehyde Prills | Fisher Scientific | 5027632 | |
New England Nuclear Blocking Agent | Perkin Elmer | 2346249 | No longer sold |
DAPI | Cell Signaling Technology | 4083S | |
Prolong Gold Antifade Mounting Media | Invitrogen | P36934 | |
Leica SPSII Spectral Confocal | Leica Biosystems | N/A | For confocal imaging |
Leica DMIL Inverted Phase Contrast Microscope | Leica Biosystems | N/A | |
Cobalt-60 Teletherapy Instrument | Atomic Energy of Canada Ltd Theratron-80 | N/A | |
Amac Box, Clear | The Container Store | 60140 | Agarose block mold |