Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Summary

The ex vivo upright droplet culture is an alternative to current in vitro and in vivo experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Abstract

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Introduction

Organ regeneration in vivo in humans is very limited; therefore, tissue engineering, the development of tissues and organs from individual cells donated by a host, is becoming an attractive potential therapy for organ replacement. However, for this therapeutic strategy to be successful, factors and cellular interactions involved in morphogenesis of the organ must be thoroughly studied and well-understood. Due to the inability to study development of specific organs with traditional approaches, researchers have turned to alternative whole embryo or whole organ cultures. Kalaskar et al.¹ have shown that ex vivo whole embryogenesis culture yields comparable results (in 58% of cultured embryos) to in utero development, suggesting that ex vivo culture methods are a feasible alternative for organogenesis studies.

An individualized organ culture system, such as this ex vivo droplet culture system, allows for whole organ analysis independent of systemic effects, while permitting manipulation of a specific signaling pathway or cellular interactions via addition of pharmacological reagents or antibodies. Traditionally, the study of fetal organ development has been limited to transgenic and knockout mouse technologies, in addition to pharmacological reagents delivered maternally. However, there are technical issues involving these techniques and treatments in vivo; most concerns revolve around the effects of influencing various organs simultaneously which often results in embryonic lethality. An additional concern of studies manipulating fetal development pharmacologically is the maternal effect of drugs on embryonic development in utero (e.g., maternal metabolism of the drug before it reaches the embryo) and if such reagents can pass through the placental barrier.

The whole organ culture technique described here was adapted from a protocol first described by Maatouk et al.², in which whole fetal gonads are incubated in ex vivo upright droplet cultures. One significant advantage of culturing fetal gonads is that small-molecule inhibitors can readily access the whole organ by simple diffusion. DeFalco et al. have shown that utilizing this ex vivo droplet culture method in conjunction with small-molecule inhibitors can be used to study signaling processes and interactions occurring during gonad development³; these processes would be difficult to examine in vivo due to technical challenges (e.g., passage of drugs through the placenta or lethality of affecting multiple organs using genetic or pharmacological approaches).

The droplet culture is not only an improvement in certain aspects over in utero experimentation, but also it is an improvement over in vitro and ex vivo systems as well. The use of cell lines to study morphogenesis is extremely difficult because they lack the diverse cell types, lack critical extracellular matrix (ECM) components that permit the formation of organ architecture, and can exhibit artifacts in signaling cascades. Although tissue engineering has made significant improvements in creating scaffolds simulating ECM, the lack of knowledge with regard to which signals are required by each cell type during organogenesis makes it challenging to build an organ system in vitro. Other ex vivo systems have been previously established to study organogenesis, or more specifically morphogenesis, and have been very successful for live imaging of fetal organs in agar⁴, transwells⁵, filters⁶, and other scaffold matrices^7,8. The advantage of the droplet culture system is that it allows the study of morphogenesis by providing the ability to utilize less reagents, which are often expensive, but also giving the organ surface tension, which is important for growth and signaling capabilities⁹.

In the mouse, initial testis morphogenesis takes place between embryonic (E) stages E11.5 and E13.5; these stages comprise the optimal time window for examining factors that influence sex-specific differentiation. Among the critical processes that occur during testis formation are the generation of testis cord architecture and the formation of a testis-specific vascular network. Utilizing this ex vivo whole organ droplet culture system, one is able to alter male-specific vascularization and inhibit testis morphogenesis through the use of a small-molecule inhibitor that blocks the activity of the receptors for vascular endothelial growth factor (VEGF); VEGF-mediated vascular remodeling is critical for testis development^10-12. This technique can successfully be applied to other organs and can target specific time windows of development. Whole-mount organ imaging allows the visualization of vital structures as well as structural and cellular changes resulting from the administration of various inhibitors. Importantly, this system is advantageous in that the researcher can bypass potential confounding effects from maternal drug administration or systemic disruption during in vivo targeted gene strategies. Thus, this whole organ ex vivo droplet culture system can significantly improve the ability to understand the interactions and signaling which occur specifically within particular organs during fetal development.

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Protocol

All mice used in these studies were CD-1 mice obtained from Charles River Laboratories. Previous culture experiments have also been performed on other strains, such as C57BL/6J (data not shown), but any strain can be used. Pregnant adult females were approximately 2-3 months old and were euthanized via CO₂ inhalation followed by cervical dislocation and bilateral thoracotomy prior to embryo removal. Mice were housed in accordance with NIH guidelines, and experimental protocols were approved by the Institutional Animal Care and Use Committee of Cincinnati Children’s Hospital Medical Center.

1. Preparation of Instruments, Culture Media, and Dishes

1. Prepare a 70% ethanol solution. Autoclave deionized water (for making humidified chambers and for making/diluting solutions). Sterilize dissection tools (forceps, scissors, and 27 G needle syringes) by spraying down with 70% ethanol.
2. Prepare 10X phosphate-buffered saline (PBS) containing calcium and magnesium (80 g NaCl, 2 g KCl, 14.4 g Na₂HPO₄, 2.4 g KH₂PO₄, 6.75 ml of 1 M CaCl₂, 3.75 ml of 1 M MgCl₂). Stir to dissolve in 1 L sterile autoclaved water and then filter with filter paper. Dilute 10-fold with water to make 1X PBS.
3. Heat inactivate fetal bovine serum (FBS) at 55 °C for 30 min and filter sterilize with a 0.22 µm syringe filter.
4. Prepare 38 ml 1X complete culture medium (called complete DMEM, cDMEM), consisting of Dulbecco’s Modified Eagle Medium (DMEM), 10% filtered heat-inactivated FBS and 1/100 dilution of penicillin-streptomycin stock. This usually should be used fresh, but can be stored at 4 °C for 1 month.
5. Reconstitute the VEGFR Tyrosine Kinase Inhibitor II (TKI II) in DMSO for a stock concentration of 5 mg/ml, and dilute stock with cDMEM to make a working solution. The final concentration for this study is 1.875 µg/ml in cDMEM. According to the company’s recommendations, stock solutions are stable for up to 6 months at -20 °C. As for working solutions, make fresh and use on the same day.
   Note: The concentration of this drug in the working solution should be two-fold higher than the desired final concentration (since it will be diluted two-fold in the droplet). At the same time, prepare the same volume of cDMEM containing the same volume of DMSO for the “control” treatment.
6. Prepare the tail digestion reagents (50 mM NaOH and 1 M Tris-HCl [pH 8.0]) separately in distilled water.
7. Make a 25 μM stock of the XY PCR primers (XY Forward 5'-TGA AGC TTT TGG CTT TGA G-3' and XY Reverse 5'-CCG CTG CCA AAT TCT TTG G-3') together in nuclease-free water.
8. Prepare 50X TAE buffer (242 g Trizma Base, 57.1 ml Glacial Acetic Acid, 100 ml 0.5 M EDTA, pH 8.5, and add water to 1 L final volume). Prepare 1X TAE Running buffer (20 ml 50X TAE diluted with water to 1 L total volume).
9. To create a 2.5% agarose solution (0.625 g agarose, 0.5 ml 50X TAE Buffer, 24.5 ml water), heat agarose solution in microwave until boiling, then let cool until about 55-65 °C (able to touch). Once cool, add 2 µl of 1% ethidium bromide solution, and pour gel.
   CAUTION! Ethidium bromide is a mutagen and teratogen; please use nitrile gloves and proper safety protocol when handling. Alternatively, other potentially less toxic gel resolving agents or dyes can be used.
10. Prepare Blocking Solution (PBS with 0.1% Triton X-100, 10% fetal bovine serum [FBS], and 3% bovine serum albumin [BSA]) for immunofluorescence.
11. Prepare Washing Solution (PBS with 0.1% Triton X-100, 1% FBS, and 3% BSA) for immunofluorescence.
12. Prepare PBTx (PBS with 0.1% Triton X-100) for immunofluorescence.
13. Prepare the incubation chambers for culture. Fill large (100 mm diameter) petri dishes with 35 ml autoclaved water. Place near where dissection and cultures will be performed.

2. Isolation of Fetal Testes from Mus musculus

1. Check the female mouse for vaginal plugs each morning. Noon on the day at which the vaginal plug is detected is considered E0.5. Euthanize the pregnant female mouse at E11.5, in a manner consistent with approved animal protocols.
2. Spray down mouse abdomen with 70% ethanol.
3. Open the belly skin and peritoneum with a V-shaped incision using fine scissors and pull back flap of tissue to expose interior organs.
4. Locate the ovaries at both ends of the uterine horn. Using scissors, cut at the ovary and the connective tissue to separate the uterus containing the embryos from the mother’s body.
5. Open the uterine wall by gently cutting the side opposite the placentae, exposing the yolk sacs. Be careful not to puncture yolk sac to avoid damaging or losing embryos.
6. Cut near the placentae to remove the embryo-containing yolk sacs and place them into a dish containing PBS with calcium and magnesium. The presence of calcium and magnesium ions will help support cell adhesion molecules to maintain tissue architecture and prevent the tissue from sticking to the dissection tools.
7. With forceps, remove the yolk sac and amnion from the embryo by carefully puncturing the yolk sac to create a hole in which the embryo can slide out. Once the embryo is outside of the yolk sac, cut the umbilical cord.
8. From this point forward, use a microscope located in a sterile tissue culture hood. Remove the head of embryo and discard by pinching with forceps on either side of neck.
9. Remove the tail and place into new microcentrifuge tube for XY PCR analysis to determine sex of embryo.
   Note: While it is optimal to culture gonads as soon as possible after harvest, it is also possible to remove tail DNA and perform XX/XY genotyping prior to culture, so that the sex of each embryo is known and only the desired sex needs to be cultured. While the genotyping is being done, embryos can be kept separated (so that they can be tracked) at 4 °C or on ice until ready for culture. One caveat is that this period outside in utero or culture conditions may potentially affect viability for culture or subsequent development during culture.
10. Secure the embryo in a supine position against the bottom of the culture dish by pinning the armpits of embryo with one pair of forceps (usually the forceps held in the weak hand). Maintain these forceps in place to hold the embryo still during the entire dissection process.
11. With other pair of forceps, remove skin covering abdomen and gently remove liver, intestines, and other organs to expose back body wall.
12. As the gonads will be located on the back body wall on either side of the dorsal aorta, a large blood vessel running along the midline of the body, scoop underneath the urogenital ridge with closed forceps and remove the urogenital ridge by opening the forceps and lifting up. As the gonads will be loosely attached to the wall, be careful not to pull up too fast without removing these connections or the gonads may stretch, causing damage to the organs.
13. Move the urogenital ridge into cDMEM in a dish to acclimate tissue to media.
14. Separate the gonad-mesonephros complex from the rest of the urogenital ridge using 27 G needles. Use one needle to cut by pressing down, and use the other to guide the tissue correctly to allow optimal separation. Avoid using a sawing motion against the dish bottom, as sharp needles will create shards of plastic that can stick to the tissue. Make sure to keep the mesonephros completely attached to the gonad.

3. Culturing of Gonad with a Small-molecule Inhibitor

1. Set two 20 µl pipettes to 15 µl each. Cut about 1-2 mm off one of the barrier pipette tips using a clean, sterilized razor blade for transfer of the gonads and addition of control cDMEM, while the other pipette will be used solely for drug-treated cDMEM.
2. Line up gonads with their long axis parallel to the pipette tip so they can easily be pipetted using the cut-off tip. Be careful of gonads sticking to the inside of the pipette tip.
3. Label one side as the control droplet and the other as the drug-containing droplet. Only two droplets can fit on a 35 mm dish lid. Make sure that labels on lids match the labels on tail-tissue-containing microcentrifuge tubes.
4. Pipette 15 µl of cDMEM containing a single gonad into a droplet in the lid of a small (35 mm) culture dish (use only the lids, as the bottoms are too tall to fit within a humidified petri dish chamber). Place two separate gonad-containing droplets on either side of the dish, well-separated from one another. Check under the microscope to ensure that the gonads have been transferred to the droplets.
5. To the droplet designated as the control, add an additional 15 µl of cDMEM containing DMSO (made in step 1.5) to make a droplet with a 30 µl total volume. Use only the “control” pipette that will not contact the drug.
6. To the other droplet (drug-treated sample), add 15 µl of TKI-II-containing cDMEM to make a droplet with a 30 µl total volume. Use only the pipette designated for drug-treated samples.
7. Using the pipette, spread out the droplets in an expanding circular pattern until they are about 15-18 mm in diameter and the gonad is located roughly in the middle. Orient the gonad so that it lies on its side and the gonad and mesonephros are easily distinguishable. Orient the lung so that the two lobes lay flat.
   1. Although droplet diameter may vary slightly, ensure that gonads are not floating and are held in place by surface tension. Make sure that droplets do not touch each other or the side of the lid. Without the surface tension, the organs will grow onto the lid during the culture and flatten, thus distorting organ morphology.
8. Carefully place the culture dish lid containing the droplets upright onto the surface of the water in the large (100 mm) humidifier dish. Ensure there are no bubbles trapped underneath the bottom of the lid and that it is lying flat on the surface of the water. Do not invert the lid and be careful not to let any water touch the top of the lid where the droplets are located.
9. To create a small, humidified chamber, immediately cover with the lid of the larger humidifier dish to enclose the gonad cultures. Act as quickly as possible to minimize any evaporation of media. Ensure that the smaller dish has room between it and the lid of the larger dish to move around freely; otherwise, condensation may make a seal and block air exchange to the tissue.
10. Once two small lids are placed into the chamber, immediately place the chamber into the incubator. Incubate the droplet cultures for 48 hr in a humidified CO₂ incubator at 37 °C.

4. Polymerase Chain Reaction for Determining the Sex of Embryos

1. Add the embryonic tails to bottom of a 1.5 ml microcentrifuge tube.
2. Add to each tube 200 µl of 50 mM NaOH.
3. Place at 95 °C for 15 min or until tissue is completely dissolved.
4. Add 50 µl of 1M Tris-HCl and vortex lightly and briefly.
5. In a 1.5 ml microcentrifuge tube, make a fresh PCR master mix by multiplying each volume by the total number of samples: 0.5 µl 25 µM Primer solution, 2.5 µl 10X Taq Buffer, 0.5 µl 10 mM dNTPs (nucleotides), 19.3 µl nuclease-free water, 0.2 µl DNA Taq polymerase. Mix well.
6. Add 23 µl of PCR master mix to PCR tubes containing 3 μl lysate of digested tails.
7. Flick tubes to mix them, then perform a short spin to bring the samples to the bottom of the tube.
8. Run XY (Short) PCR program (Table 1).
9. Load the samples (with 5x dye) and ladder in a 2.5% agarose gel in 1X TAE buffer.
10. Run the agarose gel electrophoresis until XX and XY bands can be resolved.
11. Image the gel with UV light and capture image.
12. Analyze the X-chromosome-specific vs Y chromosome-specific bands (X chromosome=331 base pairs [bp] and XY=302 bp)¹³; XY (male) samples will have two bands of 331 and 302 bp, whereas XX (female) samples will appear as a single 331 bp band.

5. Whole Mount Organ Immunofluorescence

Day 1:

1. Remove the gonads from the culture using a 1,000 µl pipette with a cut-off tip. If desired, they may be placed in a dish of PBS to wash off media and reagents. Place into PBS in a 0.5 ml microcentrifuge tube. The smaller tube size will conserve reagents and make it easier to keep track of samples in the tube.
   1. Be sure to keep control and treated samples, as well as XX and XY samples, in separate tubes and remove them from culture with separate sets of pipettes to avoid potential drug contamination.
2. Wash the gonads twice with PBS. From this point on, this protocol can use 1X PBS lacking calcium and magnesium. To wash them, allow the gonads to sink to the bottom of the tube (by gravity) and then remove the liquid above. Be careful not lose gonads by pipetting too close to them.
3. Remove as much PBS as possible from the tube. Add 250 µl of 4% paraformaldehyde in PBS containing 0.1% Triton X-100, and let incubate O/N at 4 °C on a rocker. Alternatively, this fixation can be done for 2 hr at 4 °C on a rocker. CAUTION! Paraformaldehyde is a toxic substance, so follow safety protocol when handling.

Day 2:

4. Rinse the gonads twice in 250 µl PBTx at RT.
5. Wash the gonads 3 times with 250 µl PBTx for 10 min each at RT on a rocker.
6. Block the gonads at least 1 hr in 250 µl Blocking solution at RT on a rocker.
7. Stain the gonads O/N at 4 °C on a rocker in 250 µl primary antibody diluted in Blocking Solution.

Day 3:

8. Rinse the gonads twice in 250 µl Washing Solution.
9. Wash the gonads 3 times with 250 µl Washing Solution for 10 min each at RT on a rocker.
10. Block the gonads 1 hr in 250 µl Blocking Solution at RT on a rocker.
11. Cover the microcentrifuge tube with aluminum foil to protect fluorescent secondary antibodies from light.
12. Incubate the gonads 2-4 hr at RT on a rocker in 250 µl secondary antibody diluted in Blocking Solution containing Hoechst 33342. Alternatively, perform secondary antibody and Hoechst 33342 incubation O/N at 4 °C on rocker.
13. Rinse the gonads twice in 250 µl PBTx.
14. Wash the gonads 3 times in 250 µl PBTx for 10 min each at RT on a rocker.
15. Using a cut-off pipette tip, transfer gonads onto a slide with minimal liquid. Remove excess liquid with pipette, but make sure that tissue does not dry out.
16. Orient the gonads in desired fashion with forceps, and quickly add a drop of mounting media to mount the gonads on the slide. Make sure the mounting media coats all samples completely; it is critical to avoid letting samples dry out.
17. Use coverslips (number 1.5 style) of appropriate size to gently cover samples. Let mounting media spread to contact the entire surface of coverslip. If necessary, very lightly press on the corners of the coverslip so that there are no large air bubbles.
18. Seal the slides with clear nail polish around the edges to reduce evaporation of the mounting media. Store mounted slides in the dark at 4 °C.

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Results

The ex vivo droplet culture allows one to manipulate whole organs, such as the gonad, to study cellular interactions and dynamics. Figure 1 demonstrates in a step-wise fashion how to prepare an E11.5 gonadal droplet culture. The first steps in the culture protocol include the initial removal of the embryo-containing uterus from the mother mouse (Figure 1A and 1B). After removal of the uterus from the mother, the uterine wall is cut and the embryos are liberated from the yolk sac...

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Discussion

This study demonstrates an ex vivo whole organ droplet method that has many potential applications for studying fetal development. This technique can be used for multiple organs, and allows the researcher to address biological questions that are difficult to examine using in vivo approaches due to inaccessibility of embryos and potential embryonic lethality. This culture method has additional benefits over other in vitro approaches such as mammalian cell lines: whole organs can be used, therefo...

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Materials

Name	Company	Catalog Number	Comments
Superfrost Plus Microscope Slides	Fisherbrand	12-550-15
Cover Glasses: Squares (22 mm x 22 mm, No. 1.5)	Fisherbrand	12-541B
Sally Hansen Xtreme Wear Nail Polish, Invisible	Sally Hansen
8-Strip 0.2 ml PCR Tubes & Detached Flat Caps	GeneMate	T3218-1
Pipetman L P1,000L, P200L, P20L, P10L, P2L	Gilson	FA10006M, FA10005M, FA10003M, FA10002M, FA10001M
Dumont #5 Forceps	FST	91150-20
Fine Scissors	FST	91460-11
Posi-Click 1.7 ml microcentrifuge tubes	Denville	C2170
Posi-Click 0.6 ml microcentrifuge tubes	Denville	C2176
10 μl SHARP Precision Barrier Tips	Denville	P1096FR
20 μl SHARP Precision Barrier Tips	Denville	P1121
200 μl SHARP Precision Barrier Tips	Denville	P1122
1,000 μl SHARP Precision Barrier Tips	Denville	P1126
1 ml syringe with 27 gauge needles	BD PrecisionGlide	309623
10 ml syringe	BD	305559
0.2 μM PES syringe filter	VWR	28145-501
Grade 3 Qualitative Filter Paper Standard Grade, circle, 185 mm	Whatman	1003-185
Primaria 35 mm Easy Grip Style Cell Culture Dish	Falcon/Corning	353801
Petri Dishes, Sterile (100 mm x 15 mm)	VWR	25384-088
New Brunswick Galaxy 14 S CO2 Incubator	Eppendorf	CO14S-120-0000
Biosafety Cabinet	Nuare	NU-425-400
Mini-centrifuge	Fisher Scientific	05-090-100
BioExpress GyroMixer Variable XL	GeneMate	R-3200-1XL
Mastercycler Pro Thermal Cycler with control panel	Eppendorf	950040015
SMZ445 stereomicroscope	Nikon	SMZ445
MultiImage Light Cabinet with AlphaEase Software	Alpha Innotech Corporation	Discontinued
Absolute 200 proof Ethanol	Fisher	BP2818-500
Triton X-100	Fisher	BP151-100
Sodium Phosphate (Dibasic MW 142) Na2HPO4	Fisher	S374-1
Potassium Phosphate (Monobasic MW 136) KH2PO4	Sigma-Aldrich	P5379-1KG
Sodium Chloride (NaCl)	Fisher	S671-3
Potassium Chloride (KCl)	Sigma-Aldrich	P3911-1KG
Magnesium Chloride (MgCl2)	Sigma	M2393-100g
Calcium Chloride (CaCl2)	Sigma	C5670-100g
Ambion Nuclease-Free Water	Life Technologies	AM9938
XY PCR Primer	IDT	N/A
Glacial Acetic Acid	Fisher	A38-500
Ethylenediamine Tetraacetic Acid (EDTA)	Fisher	BP2482-1
1% Ethidium bromide solution	Fisher	BP1302-10	Toxic
Agarose	GeneMate	E-3120-500
Sodium Hydroxide (NaOH)	Sigma-Aldrich	367176-2.5KG
Trizma Base	Sigma	T1503-1KG
dNTP Set, 100 mM Solutions	Thermo Scientific	R0182
DNA Choice Taq polymerase with 10x Buffer	Denville	CB-4050-3
Paraformaldehyde	Fisher	O4042-500	Toxic
FluorMount-G	Southern Biotech	0100-01
Hydrogen Chloride (HCl)	Fisher	A144212
Bovine Serum Albumin (BSA), powder, Fraction V, Heat shock isolation	Bioexpress	0332-100g
Dulbecco's Modified Eagle Medium (DMEM)	Life Technologies	11965-092
Fetal Bovine Serum (FBS), triple 100 nm filtered	Fisher	03-600-511	Heat-inactivate before using
Penicillin-Streptomycin (10,000 U/ml)	Life Technologies	15140-122	Use at 1:100
Dimethyl sulfoxide (DMSO), Hybri-max, sterile-filtered	Sigma	D2650
VEGFR Tyrosine Kinase Inhibitor II - CAS 269390-69-4 - Calbiochem	EMD Millipore	676481
Rabbit Anti-Sox9 Antibody	Millipore	AB5535	Use at dilution: 1:4,000
Rat Anti-Mouse PECAM1 (CD31) Antibody	BD Pharmingen	553370	Use at dilution: 1:250
Rabbit Cleaved Caspase-3 (Asp175) Antibody	Cell Signaling	9661S	Use at dilution: 1:250
Rat E-cadherin / CDH1 Antibody (ECCD-2)	Life Technologies	13-1900	Use at dilution: 1:500
Hoechst 3342, trihydrochloride, trihydrate	Invitrogen (Molecular Probes)	H1399	Use at 2 ug/ml
Cy3 AffiniPure Donkey Anti-Rat IgG (H+L)	Jackson Immunoresearch	712-165-153	Use at dilution: 1:500
Alexa Fluor 647 AffiniPure Donkey Anti-Rat IgG (H+L)	Jackson Immunoresearch	712-605-153	Use at dilution: 1:500
Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate	Life Technologies	A31572	Use at dilution: 1:500
Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate	Life Technologies	A21206	Use at dilution: 1:500
Donkey anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate	Life Technologies	A21208	Use at dilution: 1:500
Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate	Life Technologies	A31573	Use at dilution: 1:500