129S4 mice were a gift from Dr. Philippe Soriano, Icahn School of Medicine at Mount Sinai. This work was supported with funds from the National Institutes of Health (NIH)/National Institute of Dental and Craniofacial Research (NIDCR) R01 DE027689 and K02 DE028572 to K.A.F., F31 DE029976 to M.A.R. and F31 DE029364 to B.J.C.D.

All the procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado Anschutz Medical Campus and performed in compliance with institutional guidelines and regulations. Female 129S4 mice at 1.5-6 months of age and housed at a sub-thermoneutral temperature of 21-23 &#176;C were used for embryo harvests. A schematic workflow of the protocol is represented in Figure 1. See the Table of Materials for details regarding all materials, equipment, software, reagents, and animals used in this protocol.
1. Harvesting E11.5 mouse embryos
<ol>
	<li>Euthanize pregnant female mouse 11.5 days after detection of vaginal plug during timed mating in a CO2 chamber using IACUC-approved CO2 flow rate required for approximately 50% displacement (2.95 L/min in a 360 in3 chamber) and duration of exposure (see Table of Materials). Perform cervical dislocation as a secondary method of euthanasia. Proceed immediately to dissection.</li>
	<li>Lay the mouse body on a dissecting board with the ventral side facing up. Spray the mouse abdomen with 70% ethanol.</li>
	<li>Open the abdominal cavity by pinching and lifting the skin anterior to the vaginal opening with straight Semken forceps and cutting the lifted skin and underlying layers with straight blade surgical scissors at a 45&#176; angle on either side to generate a &#34;V&#34; shape that extends to each lateral surface roughly half-way between the forelimbs and hindlimbs.</li>
	<li>Using the Semken forceps, grip one of the uterine horns and cut below the oviduct and above the cervix with the surgical scissors. Cut away the mesometrium to allow for complete removal of the uterine horn.</li>
	<li>Transfer the dissected uterine horn to 10 mL of histology phosphate-buffered saline (PBS) [0.137 M NaCl, 2.68 mM KCl, 1.76 mM KH2PO4 monobasic, 10.14 mM Na2HPO4 dibasic, pH 7.4] in a 10 cm Petri dish.</li>
	<li>Remove the second uterine horn on the opposing side of the abdominal cavity using the same procedure described in steps 1.4-1.5.</li>
	<li>Place the 10 cm Petri dish containing both uterine horns on ice if not proceeding immediately to the dissection of individual embryos (i.e., if a second female mouse will be dissected).</li>
	<li>Under a dissecting stereo microscope, carefully dissect out each embryo from the uterine horns with Dumont #5 fine forceps. Slowly pull away the myometrium, decidua, and chorion. Tear and remove the relatively transparent amnion surrounding the embryo, and sever the umbilical cord connecting the embryo to the placenta.</li>
	<li>Transfer each dissected embryo to 2.5 mL of histology PBS in an individual well of a 12-well cell culture plate on ice using a cut plastic transfer pipet or an embryo spoon. 
	&#8203;NOTE: If working with a litter that may contain embryos of more than one genotype, save the amnion surrounding each embryo for genotyping by placing each in its own prelabeled 0.5 mL microcentrifuge tube on ice.</li>
</ol>
2. Dissecting maxillary processes from E11.5 mouse embryos
<ol>
	<li>Prepare three 10 cm Petri dishes containing 10 mL of histology PBS and keep them on ice to be used in rotation between embryos.</li>
	<li>Transfer one embryo from an individual well of the 12-well cell culture plate to one of the 10 cm Petri dishes with histology PBS on ice using a cut plastic transfer pipet or an embryo spoon.</li>
	<li>Under the dissecting microscope, separate each maxillary process from the face using the fine forceps. First, cut the anterior side of one maxillary process along the natural indentation separating the lateral nasal process and the maxillary process (Figure 2A).</li>
	<li>Second, cut the posterior side of the maxillary process along the natural indentation separating the maxillary process and the mandibular process (Figure 2A).</li>
	<li>Third, to completely separate the maxillary process, make a vertical cut from the anterior to posterior sides of the maxillary process on the eye side of the maxillary process where the natural indentations referenced above end (Figure 2A-C).</li>
	<li>Repeat these three cuts for the maxillary process on the opposing side of the face.</li>
	<li>Using a 9&#34; Pasteur pipet with a 2 mL small latex bulb, transfer the dissected pair of maxillary processes and a small droplet of approximately 30 &#181;L of histology PBS to a labeled 35 mm Petri dish on ice (Figure 2D).</li>
	<li>Follow steps 2.3-2.7 for each embryo, rotating the three 10 cm Petri dishes containing histology PBS on ice between embryos. Place individual pairs of dissected maxillary processes in separate 35 mm Petri dishes on ice. 
	NOTE: Separation of the maxillary process ectoderm and mesenchyme (steps 2.9-2.17) can be performed after dissecting the maxillary processes of all embryos in the litter. If intact maxillary processes containing both the ectoderm and mesenchyme are desired, proceed to step 2.18 below.</li>
	<li>Prepare 250 &#181;L of fresh 2% trypsin in tissue culture PBS and 250 &#181;L of 10% fetal bovine serum (FBS) in tissue culture PBS and store on ice.</li>
	<li>Place a second, small droplet of approximately 30 &#181;L of 2% trypsin in the 35 mm Petri dish separate from the first, small droplet of histology PBS containing the maxillary processes. Transfer the pair of maxillary processes to the small droplet of 2% trypsin using the Pasteur pipet (Figure 2D).</li>
	<li>Incubate the dish on ice for 15 min.</li>
	<li>Remove the 35 mm Petri dish from the ice and place under the dissecting microscope.</li>
	<li>Using the fine forceps, slowly and carefully pull off the layer of ectoderm from each maxillary process (Figure 2E). 
	NOTE: If the ectoderm is not separating from the mesenchyme in an intact sheet, incubate in 2% trypsin at room temperature (RT) for up to 5 additional min before continuing with separation. If the tissue starts to disintegrate in the 2% trypsin, move the maxillary processes to the 10% FBS as described below to neutralize the trypsin and finish the separation of the ectoderm and mesenchyme.</li>
	<li>Once the maxillary process ectoderm and mesenchyme are separated (Figure 2F), transfer the desired tissues from the pair of maxillary processes to a third, small droplet of approximately 30 &#181;L of 10% FBS - separate from the two previous small droplets - using the Pasteur pipet (Figure 2D).</li>
	<li>Place the 35 mm Petri dish on ice to stop the trypsinization.</li>
	<li>Incubate the maxillary process tissues on ice in 10% FBS for 1-2 min, and then transfer the tissues from both maxillary processes to a fourth, small droplet of approximately 30 &#181;L of prechilled histology PBS on ice - separate from the three previous small droplets - using the Pasteur pipet (Figure 2D).</li>
	<li>Incubate the maxillary process tissues in histology PBS for 1 min while gently swirling the histology PBS around the maxillary process tissues with the tip of the Pasteur pipet to ensure that all FBS is rinsed off the tissue.</li>
	<li>Transfer the pair of maxillary process tissues to a labeled 1.5 mL microcentrifuge tube on ice using the Pasteur pipet, minimizing the transfer of histology PBS. Remove any excess histology PBS in the 1.5 mL microcentrifuge tube with the Pasteur pipet.</li>
	<li>Follow the above steps to dissect the maxillary processes from each embryo in the litter.</li>
	<li>Process the samples immediately to isolate whole cell protein lysates (below) or store at -80 &#176;C long-term. Prior to long-term storage, snap-freeze the 1.5 mL microcentrifuge tube in a bath of 100% EtOH on dry ice for 5 min.</li>
</ol>
3. Isolating whole cell protein lysates from mouse maxillary processes
<ol>
	<li>If maxillary processes were previously frozen at -80 &#176;C, thaw them on ice.</li>
	<li>Add 0.1 mL of ice-cold NP-40 lysis buffer (20 mM Tris HCl pH 8, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM EDTA; stored at 4 &#176;C) with protease and phosphatase inhibitors (1x complete mini protease inhibitor cocktail [dissolved in water; stored at -20 &#176;C], 1 mM PMSF [dissolved in isopropanol; stored at 4 &#176;C], 10 mM NaF [stored at -20 &#176;C; avoid freeze/thaw], 1 mM Na3VO4 [stored at -20 &#176;C], 25 mM &#946;-glycerophosphate [stored at 4 &#176;C]) added immediately before use on ice. 
	NOTE: Cell fractionation can alternatively be performed to isolate cytoplasmic, nuclear, membrane, or mitochondrial protein fractions.</li>
	<li>Pipet up and down 10 times with a 200 &#181;L pipetman.</li>
	<li>Vortex for 10 s, and then pipet up and down 10 times with a 200 &#181;L pipetman. Avoid generating bubbles.</li>
	<li>Incubate at 4 &#176;C for 2 h while rotating end over end using a 1.5 mL/2 mL paddle with a tube revolver.</li>
	<li>Centrifuge the samples at 13,500 &#215; g for 20 min at 4 &#176;C.</li>
	<li>Collect the supernatant to a new 1.5 mL microcentrifuge tube on ice with a 200 mL pipetman.</li>
	<li>Quantify the protein concentration with the protein assay kit, using 10 &#956;L of protein lysate + 10 &#956;L of NP-40 lysis buffer for experimental samples and 3-5 dilutions of bovine serum albumin (fraction V) (BSA) in NP-40 lysis buffer at a range of 0.25-2.0 mg/mL as protein standards.</li>
	<li>Proceed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (step 5) or quickly freeze the remaining lysates on dry ice and store at -80 &#176;C long-term.</li>
</ol>
4. Isolating whole cell protein lysates from primary and/or immortalized mouse embryonic palatal mesenchyme (MEPM) cells
NOTE: Isolation and culture of primary MEPM cells from E13.5 mouse embryos and immortalized MEPM cells have been previously described32,33,34. Stimulation of cells with growth factor (steps 4.1-4.7) can be performed prior to cell lysis. If non-stimulated cells are desired, proceed to step 4.8 below.
<ol>
	<li>Aspirate the growth medium [Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 50 U/mL of penicillin, 50 &#181;g/mL of streptomycin, 2 mM L-glutamine, 10% FBS] from the MEPM cells at ~70% confluence in a 6 cm cell culture dish using a 5.75&#34; Pasteur pipet attached to a vacuum system.</li>
	<li>Wash the cells with 1 mL of tissue culture PBS.</li>
	<li>Add 3 mL of serum starvation medium [DMEM with 50 U/mL of penicillin, 50 &#181;g/mL of streptomycin, 2 mM L-glutamine, 0.1% FBS] prewarmed in a 37 &#176;C water bath.</li>
	<li>Incubate at 37 &#176;C and 5% CO2 for 23 h.</li>
	<li>Replace the serum starvation medium with 3 mL of fresh, prewarmed serum starvation medium.</li>
	<li>Incubate at 37 &#176;C and 5% CO2 for 1 h.</li>
	<li>Stimulate the cells with the growth factor of choice at an empirically-determined concentration for the desired length of time.</li>
	<li>Aspirate the medium from the MEPM cells at 80%-100% confluence using a 5.75&#34; Pasteur pipet attached to a vacuum system.</li>
	<li>Wash the cells twice with ice-cold tissue culture PBS (1 mL for a 6 cm cell culture dish); tilt the plate to the side during the last wash to ensure all the tissue culture PBS is aspirated.</li>
	<li>Lyse the cells by adding ice-cold NP-40 lysis buffer with protease and phosphatase inhibitors added immediately before use (0.1 mL for a 6 cm cell culture dish) on ice.</li>
	<li>Incubate the plate on ice for 5 min with rotation approximately every min to ensure complete coverage of the plate.</li>
	<li>Scrape the cells off the plate using a precooled cell lifter and transfer the cell suspension to a precooled 1.5 mL microcentrifuge tube on ice.</li>
	<li>Incubate the cell suspension at 4 &#176;C for 30 min while rotating end over end using a 1.5 mL/2 mL paddle with a tube revolver.</li>
	<li>Proceed with steps 3.6-3.9 described above.</li>
</ol>
5. Western blotting of whole cell protein lysates from mouse facial processes and/or MEPM cells for phosphoproteins
<ol>
	<li>Prepare whole cell protein lysate samples for SDS-PAGE.
	<ol>
		<li>Determine the amount of protein to be loaded, depending on protein abundance in the tissue/cell; 12.5 &#956;g is usually sufficient for robustly-expressed proteins in whole cell protein lysates.</li>
		<li>Add an equal volume of 2x Laemmli buffer [20% glycerol, 4% SDS, 0.004% bromophenol blue, 0.125 M Tris HCl pH 6.8] with 10% &#946;-mercaptoethanol added immediately before use. 
		CAUTION: &#946;-mercaptoethanol is skin or eye corrosive, toxic, hazardous if swallowed, and has aquatic toxicity. Handle wearing gloves, a laboratory coat, face shield, and safety glasses in a chemical fume hood. Dispose of according to Environmental Health and Safety guidelines.</li>
		<li>Mix by vortexing.</li>
		<li>Heat the samples at 100 &#176;C for 5 min in a mini dry bath.</li>
		<li>Mix by vortexing and place the samples on ice.</li>
		<li>Centrifuge at 9,400 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Place the samples briefly on ice before loading into SDS-PAGE gel, or store at -20 &#176;C long-term.</li>
	</ol>
	</li>
	<li>Perform SDS-PAGE using an electrophoresis cell with a 4%-15% precast protein gel and electrophoresis buffer35.</li>
	<li>Electrotransfer the proteins to a polyvinylidene fluoride (PVDF) membrane using transfer buffer with 0%-20% methanol (0%-10% for proteins greater than 100 kDa; 20% for proteins less than 100 kDa) added immediately before use35.</li>
	<li>Block the membrane and probe for phosphoprotein of interest.
	<ol>
		<li>Following transfer, wash the membrane in 1x tris-buffered saline (TBS) [20 mM Tris, 0.137 M NaCl, pH 7.6] for 5 min in a western blot box.</li>
		<li>Incubate the membrane in 5 mL of blocking buffer [1x TBS, 0.1% Tween 20, 5% w/v BSA; mixed well and filtered through a 25 mm syringe filter with 0.2 &#956;m pores using a 10 mL syringe with luer tip] for 1 h in a western blot box with agitation on an orbital shaker. 
		NOTE: Do not use milk as a blocking agent when characterizing phosphoproteins as it contains the phosphoprotein casein, which can cause a high, non-specific background signal.</li>
		<li>Wash the membrane three times for 5 min each in TBS-T [1x TBS, 0.1% Tween 20] in a western blot box with agitation on an orbital shaker.</li>
		<li>Incubate the membrane in 5 mL of primary antibody diluted in blocking buffer at 4 &#176;C overnight in a 50 mL conical tube using a 50 mL paddle with a tube revolver. 
		NOTE: Consult the antibody datasheet for the appropriate concentration for western blotting.</li>
		<li>Wash the membrane three times at RT for 5 min each in TBS-T in a western blot box with agitation on an orbital shaker.</li>
		<li>Incubate the membrane in 5 mL of appropriate horseradish peroxidase (HRP)-conjugated secondary antibody diluted in blocking buffer for 1 h in a western blot box with agitation on an orbital shaker.</li>
		<li>Wash the membrane three times for 5 min each in TBS-T in a western blot box with agitation on an orbital shaker.</li>
		<li>Incubate the membrane in enhanced chemiluminescence (ECL) western blotting substrate [mixing equal volumes from bottles 1 and 2; 0.5 mL of each for large (8.6 cm x 6.7 cm) membranes] for 1 min, ensuring that the entire membrane is continually exposed to the ECL western blotting substrate.</li>
		<li>Drain the membrane of excess ECL western blotting substrate and immediately develop using a chemiluminescence imager.</li>
	</ol>
	</li>
	<li>Strip the membrane and reprobe for total protein of interest.
	<ol>
		<li>Place the PVDF membrane in a transparent pouch with polyethylene lining containing 5 mL of stripping buffer [2% SDS, 62.5 mM Tris HCl&#160;pH 6.8] with 8 &#956;L/mL of &#946;-mercaptoethanol added immediately before use.</li>
		<li>Incubate at 50 &#176;C for 30 min with rocking in a hybridization oven at 11 revolutions per minute.</li>
		<li>Wash the membrane three times at RT for 5 min each in TBS-T in a western blot box with agitation on an orbital shaker.</li>
		<li>Proceed with steps 5.4.2-5.4.9 described above.</li>
	</ol>
	</li>
	<li>Quantitate western blot band densities using ImageJ software, normalizing the levels of the phosphorylated protein of interest to the levels of the total protein of interest36.</li>
</ol>

The authors have nothing to disclose.

The protocol described here allows researchers to probe critical phosphorylation-dependent signaling events during craniofacial development in a robust and reproducible manner. There are several critical steps in this protocol that ensure proper collection of data and analysis of results. Whether isolating phosphoproteins from mouse facial processes and/or cultured palatal mesenchyme cells, it is imperative to move quickly and efficiently while keeping all reagents and materials on ice when indicated. The low temperature...

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. In the mouse, this process begins at embryonic day (E) 9.5 with the formation of the frontonasal prominence and pairs of maxillary and mandibular processes, each of which contains post-migratory cranial neural crest cells. The lateral and medial nasal processes arise from the frontonasal prominence with the appearance of the nasal pits and eventually fuse to form the nostrils. Further, the medial nasal processes and maxillary processes fuse to generate the upper lip. Concurrently, palatogenesis is initiated with the formation of distinct outgrowths - the secondary palatal shelves - from the oral side of the maxillary processes at E11.5. Over time, the palatal shelves grow downward on either side of the tongue, elevate to an opposing position above the tongue, and eventually fuse at the midline to form a continuous palate that separates the nasal and oral cavities by E16.51.
These morphological changes throughout craniofacial development are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. For example, cell membrane receptors, such as subfamilies of transforming growth factor (TGF)-&#946; receptors, including bone morphogenetic protein receptors (BMPRs), and various receptor tyrosine kinase (RTK) families, are autophosphorylated upon ligand binding and activation in cranial neural crest cells2,3,4. Additionally, the G protein-coupled transmembrane receptor Smoothened becomes phosphorylated in cranial neural crest cells and craniofacial ectoderm downstream of Sonic hedgehog (SHH) ligand binding to the Patched1 receptor, resulting in Smoothened accumulation at the ciliary membrane and SHH pathway activation5. Such ligand-receptor interactions can occur through autocrine, paracrine, and/or juxtacrine signaling in craniofacial contexts. For example, BMP6 is known to signal in an autocrine manner during chondrocyte differentiation6, whereas fibroblast growth factor (FGF) 8 is expressed in the pharyngeal arch ectoderm and binds to members of the FGF family of RTKs expressed in the pharyngeal arch mesenchyme in a paracrine fashion to initiate patterning and outgrowth of the pharyngeal arches7,8,9,10. Furthermore, Notch signaling is activated in both chondrocytes and osteoblasts during craniofacial skeletal development through juxtacrine signaling when transmembrane Delta and/or Jagged ligands bind to transmembrane Notch receptors on neighboring cells, which are subsequently cleaved and phosphorylated11. However, there are other ligand and receptor pairs important for craniofacial development that have the flexibility to function in both autocrine and paracrine signaling. As an example, during murine tooth morphogenesis, platelet-derived growth factor (PDGF)-AA ligand has been demonstrated to signal in an autocrine manner to activate the RTK PDGFR&#945; in the enamel organ epithelium12. In contrast, in murine facial processes during mid-gestation, transcripts encoding the ligands PDGF-AA and PDGF-CC are expressed in the craniofacial ectoderm, while the PDGFR&#945; receptor is expressed in the underlying cranial neural crest-derived mesenchyme, resulting in paracrine signaling13,14,15,16,17. Regardless of the signaling mechanism, these receptor phosphorylation events often result in the recruitment of adaptor proteins and/or signaling molecules, which frequently become phosphorylated themselves to initiate intracellular kinase cascades such as the mitogen-activated protein kinase (MAPK) pathway18,19.
The terminal intracellular effectors of these cascades can then phosphorylate an array of substrates, such as transcription factors, RNA-binding, cytoskeletal and extracellular matrix proteins. Runx220, Hand121, Dlx3/522,23,24, Gli1-325, and Sox926 are among the transcription factors phosphorylated in the context of craniofacial development. This post-translational modification (PTM) can directly affect susceptibility to alternative PTMs, dimerization, stability, cleavage, and/or DNA-binding affinity, among other activities20,21,25,26. Additionally, the RNA-binding protein Srsf3 is phosphorylated in the context of craniofacial development, leading to its nuclear translocation27. In general, phosphorylation of RNA-binding proteins has been shown to affect their subcellular localization, protein-protein interactions, RNA binding, and/or sequence specificity28. Furthermore, phosphorylation of actomyosin can lead to cytoskeletal rearrangements throughout craniofacial development29,30, and phosphorylation of extracellular matrix proteins, such as small integrin-binding ligand N-linked glycoproteins, contributes to biomineralization during skeletal development31. Through the above and numerous other examples, it is evident that there are wide implications for protein phosphorylation during craniofacial development. Adding an additional level of regulation, protein phosphorylation is further modulated by phosphatases, which counteract kinases by removing phosphate groups.
These phosphorylation events at both the receptor and effector molecule levels are critical for the propagation of signaling pathways and ultimately result in changes in gene expression in the nucleus, driving specific cell activities, such as migration, proliferation, survival, and differentiation, which result in proper formation of the mammalian face. Given the context specificity of protein interactions with kinases and phosphatases, the resulting changes in PTMs, and their effects on cell activity, it is critical that these parameters be studied in a physiologically-relevant setting to gain complete understanding of the contribution of phosphorylation events to craniofacial development. Here, examples of two contexts in which to study phosphorylation of proteins and, thus, activation of signaling pathways during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves - both primary32 and immortalized33. At E11.5, the maxillary processes are in the process of fusing with the lateral and medial nasal processes1, thereby representing a critical timepoint during mouse craniofacial development. Further, maxillary processes and cells derived from the palatal shelves were chosen here because the latter structures are derivatives of the former, thereby providing researchers the opportunity to interrogate protein phosphorylation in vivo and in vitro in related contexts. However, this protocol is also applicable to alternative facial processes and developmental timepoints.
A critical problem in studying phosphorylated proteins is that they are easily dephosphorylated during protein isolation by abundant environmental phosphatases. To overcome this barrier, adaptations and modifications to standard laboratory methods that allow for isolation of phosphorylated proteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphorylated proteins. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various signaling pathways active during craniofacial development.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Block for mini dry bath</td>
			<td>Research Products International Corp</td>
			<td>400783</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc XRS+ imaging system with Image Lab software</td>
			<td>Bio-Rad</td>
			<td>1708265</td>
			<td>chemiluminescence imager</td>
		</tr>
		<tr>
			<td>CO2 incubator, air jacket</td>
			<td>VWR</td>
			<td>10810-902</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting board, 11 x 13 in</td>
			<td>Fisher Scientific</td>
			<td>09 002 12</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis cell, 4-gel, for mini precast gels with mini trans-blot module</td>
			<td>Bio-Rad</td>
			<td>1658030</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Fisher Scientific</td>
			<td>UVP95003001</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge 5415 D with F45-24-11 rotor (Eppendorf)</td>
			<td>Sigma Aldrich</td>
			<td>Z604062</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini dry bath</td>
			<td>Research Products International Corp</td>
			<td>400780</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>VWR</td>
			<td>89032-092</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>VWR</td>
			<td>89231-662</td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply for SDS-PAGE</td>
			<td>Bio-Rad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangular ice pan, maxi 9 L</td>
			<td>Fisher Scientific</td>
			<td>07-210-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemi 508 stereo microscope with stand K LAB, LED ring light</td>
			<td>Zeiss</td>
			<td>4350649020000000</td>
			<td>dissecting microscope</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>VWR</td>
			<td>62344-641</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube revolver</td>
			<td>Fisher Scientific</td>
			<td>11 676 341</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Fisher Scientific</td>
			<td>02 215 414</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>VWR</td>
			<td>89501-472</td>
			<td></td>
		</tr>
		<tr>
			<td>Western blot box</td>
			<td>Fisher Scientific</td>
			<td>NC9358182</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dishes, 6 cm</td>
			<td>Fisher Scientific</td>
			<td>12-565-95</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates, 12 well</td>
			<td>Fisher Scientific</td>
			<td>07-200-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lifters</td>
			<td>Fisher Scientific</td>
			<td>08-100-240</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2</td>
			<td>Airgas</td>
			<td>CD USP50</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes, polypropylene, 50 mL</td>
			<td>Fisher Scientific</td>
			<td>05-539-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo spoon</td>
			<td>Fine Science Tools</td>
			<td>10370-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 0.5 mL</td>
			<td>VWR</td>
			<td>89000-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 1.5 mL</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipet, 5.75&#34;</td>
			<td>Fisher Scientific</td>
			<td>13-678-6A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipet, 9&#34;</td>
			<td>VWR</td>
			<td>14672-380</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, 10 cm</td>
			<td>Fisher Scientific</td>
			<td>08-757-100D</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, 35 mm</td>
			<td>Fisher Scientific</td>
			<td>FB0875711YZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Pouches, transparent, polyethylene lining</td>
			<td>Fisher Scientific</td>
			<td>01-812-25B</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Fisher Scientific</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Semken forceps</td>
			<td>Fine Science Tools</td>
			<td>11008-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Small latex bulb, 2 mL</td>
			<td>VWR</td>
			<td>82024-554</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter, 25 mm, 0.2 &#956;m pore size</td>
			<td>Fisher Scientific</td>
			<td>09-740-108</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe with luer tip, 10 mL</td>
			<td>VWR</td>
			<td>BD309604</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipet</td>
			<td>Fisher Scientific</td>
			<td>13-711-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Western blot cassette opening lever</td>
			<td>Bio-Rad</td>
			<td>4560000</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatmann 3MM chr chromatography paper</td>
			<td>Fisher Scientific</td>
			<td>05-714-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-15% Precast protein gels, 10-well, 30 &#181;L</td>
			<td>Bio-Rad</td>
			<td>4561083</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate disodium salt hydrate</td>
			<td>Sigma Aldrich</td>
			<td>G5422-25G</td>
			<td>stock concentration 1 M</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M3148-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin, fraction V, heat shock tested</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Fisher Scientific</td>
			<td>AC403140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete mini protease inhibitor cocktail</td>
			<td>Sigma Aldrich</td>
			<td>11836153001</td>
			<td>stock concentration 25x</td>
		</tr>
		<tr>
			<td>DC protein assay kit II</td>
			<td>Bio-Rad</td>
			<td>500-0112</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>E7, mouse monoclonal beta tubulin primary antibody, concentrate 0.1 mL</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>E7</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>ECL western blotting substrate</td>
			<td>Fisher Scientific</td>
			<td>PI32106</td>
			<td>low picogram range</td>
		</tr>
		<tr>
			<td>ECL western blotting substrate</td>
			<td>Genesee Scientific</td>
			<td>20-302B</td>
			<td>low femtogram range</td>
		</tr>
		<tr>
			<td>Electrophoresis buffer, 5 L</td>
			<td>Bio-Rad</td>
			<td>1610772</td>
			<td>stock concentration 10x</td>
		</tr>
		<tr>
			<td>Ethanol, 200 proof, 1 gallon</td>
			<td>Decon Laboratories, Inc.</td>
			<td>2705HC</td>
			<td>EtOH</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid, Di Na salt dihydr. (crystalline powd./electrophor.)</td>
			<td>Fisher Scientific</td>
			<td>BP120-500</td>
			<td>EDTA</td>
		</tr>
		<tr>
			<td>Fetal bovine serum, characterized, US origin, 500 mL</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol (certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>G33-4</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-conjugated secondary antibody, goat anti-mouse IgG</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>115-035-146</td>
			<td>1:20,000</td>
		</tr>
		<tr>
			<td>HRP-conjugated secondary antibody, goat anti-rabbit IgG</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>111-035-003</td>
			<td>1:20,000</td>
		</tr>
		<tr>
			<td>Hydrochloric acid solution, 6N (certified)</td>
			<td>Fisher Scientific</td>
			<td>SA56-500</td>
			<td>HCl</td>
		</tr>
		<tr>
			<td>Igepal Ca - 630 non-ionic detergent</td>
			<td>Fisher Scientific</td>
			<td>ICN19859650</td>
			<td>Nonidet P-40</td>
		</tr>
		<tr>
			<td>Isopropanol (HPLC)</td>
			<td>Fisher Scientific</td>
			<td>A451-1</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td>stock concentration 200 mM</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A454-4</td>
			<td></td>
		</tr>
		<tr>
			<td>p44/42 MAPK (Erk1/2) primary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9102S</td>
			<td>1:1,000; anti-Erk1/2</td>
		</tr>
		<tr>
			<td>PDGF-BB recombinant ligand, rat</td>
			<td>Fisher Scientific</td>
			<td>520BB050</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF Receptor &#946; primary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>3169S</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>stock concentration 100 U/mL, 100 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride, 99%</td>
			<td>Fisher Scientific</td>
			<td>AC215740100</td>
			<td>PMSF; stock concentration 100 mM</td>
		</tr>
		<tr>
			<td>Phospho-p44/42 MAPK (Erk1/2) primary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9101S</td>
			<td>1:1,000, anti-phospho-Erk1/2</td>
		</tr>
		<tr>
			<td>Phospho-PDGF Receptor &#945; /PDGF Receptor &#946; primary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>3170S</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Potassium chloride (white crystals)</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td>KCl</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (white crystals)</td>
			<td>Fisher Scientific</td>
			<td>BP362-500</td>
			<td>KH2PO4</td>
		</tr>
		<tr>
			<td>SDS solution, 10%</td>
			<td>Bio-Rad</td>
			<td>161-0416</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (crystalline/biological,certified)</td>
			<td>Fisher Scientific</td>
			<td>S671-3</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Sodium fluoride (powder/certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>S299-100</td>
			<td>NaF; aliquot for one time use; stock concentration 1 M</td>
		</tr>
		<tr>
			<td>Sodium orthovanadate, 99%</td>
			<td>Fisher Scientific</td>
			<td>AC205330500</td>
			<td>Na3VO4; stock concentration 100 mM</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic anhydrous (granular or powder/certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td>Na2HPO4</td>
		</tr>
		<tr>
			<td>Tissue culture PBS</td>
			<td>Fisher Scientific</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer buffer, 5 L</td>
			<td>Bio-Rad</td>
			<td>1610771</td>
			<td>stock concentration 10x</td>
		</tr>
		<tr>
			<td>Tris base (white crystals or crystalline powder/molecular biology)</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>BioWorld</td>
			<td>21560033</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Western blot molecular weight marker</td>
			<td>Bio-Rad</td>
			<td>1610374</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Female 129S4 mice</td>
			<td></td>
			<td></td>
			<td>gift of Dr. Philippe Soriano, Icahn School of Medicine at Mount Sinai</td>
		</tr>
	</tbody>
</table>

isolation of whole cell protein lysates from mouse facial processes and cultured palatal mesenchyme cells for phosphoprotein analysis

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.

When attempting to characterize the phosphorylation of proteins isolated from mouse facial processes and/or cultured palatal mesenchyme cells, the representative results will ideally reveal a distinct, reproducible band following western blotting with an anti-phosphoprotein antibody that runs at or near the height of the corresponding total protein band (Figure 3). However, if extensive phosphorylation of the protein occurs, there may be a slight upward shift of the phosphoprotein band compa...

Watch this Scientific Journal Video about Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis at JoVE.com

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal ...

isolation-whole-cell-protein-lysates-from-mouse-facial-processes

Research

JoVE Journal

Developmental Biology

1.8K Views.  University of Colorado Anschutz Medical Campus. The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

Video: Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...