Name: dissection and explant culture of murine allantois for the in vitro analysis of allantoic attachment
Uploaded: 2018-01-13T13:00:00
Description: Watch this Scientific Journal Video about Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment at JoVE.com

This work was supported by the Deutsche Forschungsgemeinschaft SFB688 (to A.G.).

Mouse breeding was approved by the Regierung Unterfranken, and all analyses were carried out in strict accordance with all German and European Union applicable laws and regulations concerning care and use of laboratory animals.
1. Coating of Microtiter Plates with &#945;4&#946;1 Integrin for Ex Utero Allantois Explant Culture
<ol>
	<li>Reconstitute the lyophilized murine &#945;4&#946;1 integrin at 200 &#181;g/mL in sterile phosphate-buffered saline (PBS). Dilute to a final concentra...

<ol>
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E., Downs, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+murine+allantois:+emerging+paradigms+in+development+of+the+mammalian+umbilical+cord+and+its+relation+to+the+fetus.">The murine allantois: emerging paradigms in development of the mammalian umbilical cord and its relation to the fetus.</a> Genesis. 45 (5), 237-258 (2007).</li><li>Arora, R., Papaioannou, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+murine+allantois:+a+model+system+for+the+study+of+blood+vessel+formation.">The murine allantois: a model system for the study of blood vessel formation.</a> Blood. 120 (13), 2562-2572 (2012).</li><li>Watson, E. D., Cross, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+structures+and+transport+functions+in+the+mouse+placenta.">Development of structures and transport functions in the mouse placenta.</a> Physiology (Bethesda). 20, 180-193 (2005).</li><li>Yang, J. T., Rayburn, H., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+events+mediated+by+alpha+4+integrins+are+essential+in+placental+and+cardiac+development.">Cell adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development.</a> Development. 121 (2), 549-560 (1995).</li><li>Gurtner, G. 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M., Temkin, R., Gifford, S., McHugh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+murine+allantois+by+allantoic+explants.">Study of the murine allantois by allantoic explants.</a> Dev Biol. 233 (2), 347-364 (2001).</li><li>Hou, W., Sarikaya, D. P., Jerome-Majewska, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+culture+of+pre-placental+tissues+reveals+that+the+allantois+is+required+for+maintained+expression+of+Gcm1+and+Tpbpalpha.">Ex vivo culture of pre-placental tissues reveals that the allantois is required for maintained expression of Gcm1 and Tpbpalpha.</a> Placenta. 47, 12-23 (2016).</li><li>Zeigler, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+allantois+and+chorion,+when+isolated+before+circulation+or+chorio-allantoic+fusion,+have+hematopoietic+potential.">The allantois and chorion, when isolated before circulation or chorio-allantoic fusion, have hematopoietic potential.</a> Development. 133 (21), 4183-4192 (2006).</li><li>Zaidel-Bar, R., Itzkovitz, S., Ma'ayan, A., Iyengar, R., Geiger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+atlas+of+the+integrin+adhesome.">Functional atlas of the integrin adhesome.</a> Nature cell biology. 9 (8), 858-867 (2007).</li></ol>

In the presence of serum, which is a rich source of pro-adhesive extracellular matrix molecules such as fibronectin, allantois explants will readily attach to various plastic, glass, or filter surfaces9,16. Thus, if the aim of the performed assay is to specifically investigate the effect of a genetic modification or any other treatment on allantois attachment to immobilized &#945;4&#946;1, it is critical to block non-specific binding sites (e.g., with bo...

The placenta is indispensable for embryonic growth and development in the uterine environment. It constitutes the interface for oxygen, metabolite, and nutrient exchange between the fetal and maternal circulation, and also functions as an endocrine and immunological organ. Any endogenous or exogenous insult that impairs placental development can lead to intrauterine growth retardation, fetal loss, or pregnancy complications, both in mice and in humans1.While the number of targeted mouse mutations that cause abnormal placental physiology continues to increase2, with 219 genotypes currently listed on the Mouse Genome Informatics (MGI) website (http://www.informatics.jax.org/vocab/ mp_ontology/MP:0010038), many of these phenotypes are not well understood from a mechanistic point of view. In addition to genetic alterations, small molecule drugs, monoclonal antibodies, toxins, pathogens, excess metabolites, or various environmental agents may affect placenta development and function3,4,5,6,7. Yet, simple in vitro assays that model critical steps in placenta development are scarce.
Murine placental development is initiated in early midgestation, when the allantois (the developmental precursor of the umbilical cord) emerges from the posterior end of the embryo as a bud that grows toward the chorion8. Chorioadhesive cells in the outer layer of the allantoic bud mediate the attachment and spreading of the allantois on the surface of the chorion (chorioallantoic &#34;fusion&#34;). The chorion subsequently folds into villi into which the vasculature of the allantois grows to form the labyrinth, the inner placental layer where nutrients and oxygen are exchanged between closely juxtaposed maternal blood sinusoids and embryonic vessels9,10.
The attachment of the allantois to the chorion is the initial and critical step in labyrinth formation, and defects in this process are among the most common causes of embryonic lethality in midgestation1. Although a number of mouse mutants have been described where chorioallantoic attachment fails to occur10, and the MGI database currently lists 108 mouse mutants that are characterized by abnormal chorioallantoic fusion (http://www.informatics.jax.org/vocab/mp_ontology/MP:0002824), the intercellular interaction between the vascular cell adhesion molecule-1 (VCAM-1, expressed on the allantoic mesoderm) and &#945;4&#946;1 integrin (expressed on the chorionic mesothelium, which is derived from extraembryonic mesoderm) appears to be indispensable for chorioallantoic attachment and fusion11,12,13. Immunohistochemical staining of whole-mount wild-type embryos has shown that VCAM-1 is expressed within the distal two-thirds of the allantoic stalk at approximately embryonic day (E) 7.513 (1-4 somite stage), and that its expression remains high during chorioallantoic attachment and -fusion11,12. Failure of allantoic attachment to the chorion is typically detected by histological analyses of uterus buds. However, allantois sizes are physiologically highly variable in between and even within litters of the same developmental stage14, and the allantois can only attach to the chorion when it has reached a sufficient size to make physical contact. Reflecting this natural variability, chorioallantoic attachment takes place sometime between ~ E 8.0 and E 9.0 in utero, and a statistically reliable evaluation of this process by histology is therefore dependent on the analysis of a large number of conceptuses to obtain enough specimens at the appropriate developmental stage.
Here, we describe a method to assess allantoic attachment ex utero that is less dependent on allantois size. We demonstrate the dissection of mouse embryos and their allantoides from the uteri of pregnant mice ~ 8 days post coitum (dpc; dpc 0.5 designates the detection of a vaginal plug), and the subsequent culture of allantois explants on immobilized &#945;4&#946;1 integrins. This method enables a rapid, functional evaluation of the attachment and spreading behavior of multiple allantois explants in parallel and at different successive time points. The protocol may be used to screen for the effects of targeted mutations, drugs, or various environmental factors on allantois attachment ex vivo.

<table border="0" cellpadding="0" cellspacing="0" width="1607">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:319px;">C57BL/6J wildtype mice</td>
			<td style="width:256px;">The Jackson Laboratory</td>
			<td style="width:119px;">Stock No:&#160;000664</td>
			<td style="width:915px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 % (v/v) Ethanol</td>
			<td style="width:256px;">VWR International</td>
			<td>930031006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard pattern forceps</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>11000-12</td>
			<td style="width:915px;">Forceps used to open the abdominal cavity.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Micro dissecting forceps</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td style="width:119px;">11254-20</td>
			<td>Dumont # 5 medical biology forcepts with fine, sharp tip.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Fine scissors&#160;</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>14094-11</td>
			<td>Straight blades.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring scissors&#160;</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>15012-12</td>
			<td>Noyes spring scissors with 14 mm blades for the dissection of uterus buds.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microspoon</td>
			<td style="width:256px;">Carl Roth</td>
			<td>AT18.1</td>
			<td>5 mm Spoon diameter.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microtiter plates</td>
			<td style="width:256px;">ibidi</td>
			<td style="width:119px;">81501</td>
			<td>We use uncoated, sterile angiogenesis slides (internal volume 50 &#181;L) with a hydrophobic surface that is not tissue-culture treated to reduce non-a4b1-mediated binding to plastic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">10 cm dish</td>
			<td style="width:256px;">Nunc</td>
			<td style="width:119px;">150350</td>
			<td>Sterile tissue culture dishes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">3.5 cm dish</td>
			<td style="width:256px;">Nunc</td>
			<td style="width:119px;">150288</td>
			<td>Sterile tissue culture dishes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Large orifice pipette tips&#160;</td>
			<td style="width:256px;">Biozym</td>
			<td style="width:119px;">VT0140X</td>
			<td>Low binding pipette tips, 200 &#181;L.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">a4b1 integrin</td>
			<td style="width:256px;">R&#38;D Systems</td>
			<td style="width:119px;">6054-A4</td>
			<td>Murine a4b1 integrin recombinantly expressed and purified from a Chinese hamster ovary cell line.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:319px;">Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td>Without Ca2+ and Mg2+.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:319px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td style="width:256px;">PAN Biotech</td>
			<td style="width:119px;">P04-03600</td>
			<td style="width:915px;">The formulation contains 4.5 g/L glucose, 110 mg/L sodium pyruvate and 3.7 g/L NaHCO3 but no L-glutamine, which is added separately.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:319px;">Penicillin/Streptomycin</td>
			<td style="width:256px;">Gibco (ThermoFisher Scientific)</td>
			<td style="width:119px;">15140-122</td>
			<td>100 x (10,000 U/mL Penicillin and 10,000 &#181;g/mL streptomycin) stock solution.&#160;Prepare and store aliquots at -20 &#176;C to avoid freeze/thaw.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">L-Glutamine</td>
			<td style="width:256px;">PAN Biotech</td>
			<td style="width:119px;">P04-80100</td>
			<td>100 x (200 mM) Stock solution. Prepare and store aliquots at -20 &#176;C to avoid freeze/thaw.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Fetal bovine serum&#160;</td>
			<td style="width:256px;">Biochrom</td>
			<td style="width:119px;">S 0115</td>
			<td>We use heat-inactivated FBS (heated to 56 &#176;C for 30 min with mixing to inactivate complement).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Bovine serum albumin (BSA)</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">A7030</td>
			<td>We use protease- and fatty acid-free albumin. Prepare a 0.1 % (w/v) solution in DMEM. Sterile filter the solution through a disposable cell culture filter with a 0.22 &#181;m pore size and low protein-binding membrane, and store aliquots at -20 &#176;C.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">para-Formaldehyde</td>
			<td style="width:256px;">Carl Roth</td>
			<td>0335.2</td>
			<td style="width:915px;">To make a formaldehyde solution in PBS, weigh para-formaldehyde powder in a ventilated hood, and add it to PBS preheated to ca. 60 &#176;C in a beaker on a stir plate. Add 1 N NaOH dropwise until solution clears. Let solution cool to room temperature, and filter through 0.22 mm filter syringe. Adjust pH with diluted HCl to ca. 6.9, and adjust to final volume with PBS. We aliquot and freeze the solution, but it can also be stored at 4 &#176;C for approximately four weeks.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Triton X-100</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">X100</td>
			<td>Use wide-orifice pipette tips to handle undiluted Triton X-100.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Normal goat serum</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td>G9023</td>
			<td>To block non-specific binding of antibodies in immunofluorescence analyses.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">a-CD31 Antibodies</td>
			<td style="width:256px;">BD Biosciences</td>
			<td>550274</td>
			<td>Purified rat anti-mouse monoclonal antibodies, clone MEC 13.3.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary goat anti-rat antibodies</td>
			<td style="width:256px;">Thermo Fisher</td>
			<td>A-11006</td>
			<td>Goat anti-rat IgG (heavy and light chains), cross-adsorbed, fluorescently labeled with Alexa Fluor 488. Other fluorescent labels are also possible.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Mowiol 4-88</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td>81381</td>
			<td>Mounting medium for cytochemistry. MW ~31,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Labovert&#160;</td>
			<td style="width:256px;">Leitz</td>
			<td style="width:119px;">Labovert&#160;</td>
			<td>Inverted phase contrast microscope equipped with a 4 x and 10 x objective for somite pair counting. Other phase contrast microscopes (such as those that are routinely used in tissue culture) are also suitable.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">M80</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">M80</td>
			<td>Stereomicroscope with 8 : 1 zoom range (yielding a magnification between 7.5 x and 60 x) for embryo and allantois dissection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">DM4000B</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">DM4000B</td>
			<td>Microscope system for histology with LED illumination. We use HCX PL FLUOTAR 5 x/0.15 and HC PL FLUOTAR 10 x/0.30 objectives. Other microscopes equipped phase contrast and fluorescence optics can be used to score allantois attachment.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Digital camera</td>
			<td style="width:256px;">JVC</td>
			<td style="width:119px;">KY-F75U</td>
			<td>3-CCD digital capture camera attached to the DM4000B LED microscope. We use Diskus software (Hilgers) to operate both the microscope and the camera.</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:319px;">Confocal microscope</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">SP5</td>
			<td style="width:915px;">Confocal microscope for higher-resolution imaging of endothelia in the vascular plexus of allantois explants. Immunofluorescence images were taken with a 40 x objective.</td>
		</tr>
	</tbody>
</table>

dissection and explant culture of murine allantois for the in vitro analysis of allantoic attachment

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental insults that disturb placenta development or function can cause early pregnancy loss in mice and humans. Nevertheless, simple in vitro assays to screen for potential effects on placenta formation are lacking. Here, we focus on modeling the first and critical step in placenta formation, which consists of the attachment of the allantois to the chorion. We describe a method to rapidly assess the attachment of allantoic explants on immobilized &#945;4&#946;1 integrin, which serves as a chorio-mimetic substrate.This in vitro approach enables a qualitative evaluation of the attachment and spreading behavior of multiple allantois explants at different consecutive time points. The protocol may be used to investigate the effect of targeted mouse mutations, drugs, or various environmental factors that have been linked to pregnancy complications or fetal loss on allantois attachment ex vivo.

This protocol describes a method to isolate and explant murine allantoides ex vivo, and details the qualitative assessment of allantois attachment to &#945;4&#946;1 integrin, a critical process during in vivo chorioallantoic fusion. The representative results shown in Figure 1A-H demonstrate the successive steps of allantois isolation starting from the pregnant uterus. Maternal tissues that surround the uter...

Watch this Scientific Journal Video about Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment at JoVE.com

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

We describe an in vitro assay to model chorioallantoic attachment, the first step in placenta formation. The protocol demonstrates the dissection and explant culture of murine allantoides on immobilized &#945;4&#946;1 integrin. Allantois attachment is evaluated microscopically at pre-determined time points.

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

The overall goal of this procedure is the dissection and explant culture of murine allantois for the analysis of allantoic attachment in vitro. This method can help answer key questions in the field of mammalian embryo development, such as the control of allantoic attachment for placenta formation. The main advantage of this technique is that it models the first and critical step in placenta formation in vitro.Demonstrating the procedure will be Kerstin Hadamek, a Technician from my laboratory. To set up the plates, first add 20 microliters of freshly made Alpha 4/Beta 1 integrin solution into each well. Plan for one allantois per well.Do not deposit any solution with air bubbles, as this will prevent proper coating. After loading the wells, incubate the plate for an hour in a humidified incubator. After the incubation, aspirate the supernatant from the wells while holding the plate at an incline, so the pipette can gently suck out the solution without touching the well bottoms.Next, wash the wells twice using 50 microliters of warm culture medium. When removing the washing medium, use gentle suction. Apply 50 microliters of block solution to each well.Then, incubate the plate for at least 30 minutes until they are needed. Allantois explants are very sticky. So, the block step is critical for the success of the protocol.After sacrificing a pregnant mouse, disinfect the abdominal area with 70%ethanol. Next, open the abdominal skin by making a small incision at the midline with scissors. Use gloved fingers to hold the skin above and below the incision, and pull the skin apart towards the chest and tail of the mouse.Now, grasp the peritoneum with forceps and with the other hand, make a V-shaped incision from the anterior towards the posterior. Displace the tissues to access the abdominal cavity, and then push the intestines to the side to access the uterus. Now, locate the two uterine horns.While holding the cervix with forceps, which is the caudal segment of the uterine horn, use fine scissors to cut the ligaments. Then, detach both uterine horns by severing the oviducts with scissors. Once detached, use forceps to transfer the uterus into a 10 centimeter dish containing room temperature PBS.In the dish, remove the adipose tissue, vessels, and nerves surrounding the uterine horns using fine forceps and spring scissors. Then, proceed with the fine dissection of the embryos. Perform the embryo dissection under a stereo microscope that has an eight to one zoom range.Begin with separating the embryos by cutting in between the implantation sites with scissors. Grasp the muscular uterine lining of the uterus buds with forceps and pull it apart to expose the decidua. Next, use forceps to transfer the uterus buds into a new, 10 centimeter dish with PBS.Now, secure the decidua around the embryo with forceps. The mesometrial pull of the decidua is highly vascularized, and can be wider at its base than the anti-mesometrial region containing the embryo. Now, remove the mesometrial part of the decidua.Make an incision on the anti-mesometrial side of the decidua with fine forceps, and peel the decidua away with forceps to expose the embryo. Isolate the embryo using a second pair of forceps. Using a micro spoon, collect the embryo.Deposit the embryo into a drop of PBS in a 3.5 centimeter dish. Then within the dish, transfer into a second drop of PBS. Now, dissect the embryo from the yolk sac using forceps.Because maternal tissue can contaminate the yolk sac preparation and lead to erroneous genotyping, dissect the parietal yolk sac, including Reichert's membrane and the ectoplacental cone. Now, transfer the isolated yolk sac along with some PBS into a storage tube for molecular analysis. Store these samples at negative 20 degrees Celsius.The next step in the dissection is to cut the allantois from its basal insertion site at the posterior end of the embryo. This requires the use of fine forceps. If developmental staging is required, count the somite pairs of the embryo under phase-contrast optics.The chorio-allantoic attachment and fusion occur from E8 to E9, and involves at least six somite pairs. For the allantoides, load the pre-coated plate wells with 40 microliters of DMEM supplemented with fetal bovine serum, L-glutamine, and antibiotics. Then, collect each allantois with about five microliters of PBS using a 200 microliter pipette tip with a wide orifice.Deposit each into a prepared plate well. Now, incubate the allantoides to the desired time point. Do not move the plate excessively, as the goal is to study their attachment.At each time point, score their attachment using phase-contrast or differential interference contrast optics. Begin with fully spread explants that have been cultured for 20 to 24 hours. Aspirate the culture medium and add 40 microliters of paraformaldehyde to each well.After 10 minutes of fixation, aspirate the fixative and wash the tissues three times. For each wash, apply 40 microliters of PBS, wait five minutes, and remove the PBS with gentle suction. Next, permeablize the cells and block non-specific binding sites by adding 40 microliters of PBS, supplemented with detergent and serums.Let the solution incubate with the tissues for 30 minutes at room temperature. Next, wash out the solution with three applications of PBS. Then, apply 20 microliters of staining solution, containing the anti-CD31 primary antibody.Conduct the stain overnight at four degrees Celsius. The following morning, wash the tissues three times using PBS. Next, apply 20 microliters of the secondary antibody solution, and incubate the tissues for an hour at room temperature protected from light.After washing out the antibodies, as before, add 20 microliters of mounting medium to each well. Shield the plate from light and in about one hour, the medium will solidify. Then, save the plates at four degrees Celsius until they can be visualized.Ex-utero, the distal tip of allantoides isolated from wild type C57 block 6J mice were often attached by six hours. Full attachment of the entire allantois was always observed within 12 hours. There was a diversity of allantois explant and attachment morphologies.By 24 hours, a CD31-positive vascular plexus was observable in the flattened, circular explants. Thus, this protocol provides a culturing method that also enables the differentiation of allantois explants and at the study of the blood vessel formation in vitro. Once mastered, allantois dissection can be done in less than two hours, if it is performed properly.The evaluation of allantois explant attachment can be performed within a few minutes on a microscope. Following this procedure, gene trail we did mice, small molecule drugs, therapeutic antibodies. Toxins or pathogens can be screened for.Potential adverse effects on placenta development.

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