This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.
Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.
The human endometrium is the inner lining of the uterus and undergoes monthly cycles of breakdown and repair as a preparation for possible pregnancies. It consists of a single layer of epithelial cells lining the uterine lumen and the underlying stroma which varies in thickness according to fluctuations of ovarian hormones estrogen and progesterone. During the luteal phase, the postovulatory progesterone surge will induce differentiation of endometrial stromal cells into larger, round decidual cells, a process called decidualization1,2. In human, this phenomenon occurs spontaneously to form the predecidua, but becomes more pronounced upon embryo implantation. A functional consequence of decidualization is that the uterus transiently becomes receptive to embryo implantation. This interval is labeled as the 'window of implantation'. During the early period of embryo implantation, the decidualized endometrial stromal cells surrounding the implanting embryo will provide a barrier to prevent the passage of harmful substances to the embryo3. During pregnancy, this decidua will provide nutrients for the developing fetus before placentation has taken place, and will later form the maternal part of the placenta4.
Ethical and practical considerations limit the design of human studies to investigate the process of decidualization and have prompted the use of animal models. Being a member of the hemochorial placentation group, the endometrium of rodents will also undergo decidualization prior to placentation5. In rodents, however, the initiation of the decidualization process depends on the presence of blastocysts in the uterine lumen, indicating that an extra stimulus is necessary to trigger the decidual responses6. This implies an intricate communication between the endometrial epithelial cells that have direct contact with the blastocyst, and the underlying stromal cells. Nevertheless, decidualization can be artificially induced using stimuli as mechanical scratching or the instillation of oil in a hormonally primed uterus. Although in vivo studies using artificial decidualization models have allowed us to improve our insights in the complex signaling process of decidualization, mechanistic in-depth studies, e.g., investigation of the indispensable communication between epithelial and stromal cells, are limited. Therefore, in vitro decidualization studies can be used to manipulate important pathways and study fundamental processes. To this end, primary endometrial stromal cell cultures can be set up from human or rodent endometrium. Employing rodents consents the use of genetically modified animals to investigate the role of a specific protein of interest during the decidualization process. However, immature, prepuberty mice are often used in order to avoid complications of the estrous cycle, while in other cases uteri are collected from all phases of the estrous cycle, which results in a heterogeneity in the cultures. Moreover, the process of decidualization has been studied in different protocols, e.g., by (i) supplementing hormones (estrogen, progesterone or cyclic adenosine monophosphate (cAMP)) to the culturing medium, or by (ii) isolation of decidual cells from mice at day 4.5 of (pseudo)pregnancy, which demand additional need for plug checking and vasectomized males. These limitations demonstrate the necessity of a standardized method to study in vitro decidualization. In this study, a physiological model to examine in vitro decidualization starting from adult, non(pseudo)pregnant mice will be presented. In this method, daily injection of 17β-estradiol (E2) will induce proliferation of endometrial cells, resulting in an increased yield of homogenous and pure cell populations. Furthermore, the use of a coculturing system allows the study of the epithelial-stromal crosstalk and the ability to manipulate the process of decidualization on different levels.
AlexaFluor
All animal experiments for this study were approved by the ethical review committee of the KU Leuven (Belgium) (Project P174/2013). Mice were housed under standard conditions, with ad libitum access to food pellets and tap water, and kept under controlled conditions (23 ±1.5 °C, relative humidity 40 – 60%, 12/12 light/dark cycle).
1. Mice
2. Preparations
3. Preparations Required 24 h before Isolation
4. Isolation and Culture of Mouse Endometrial Epithelial Cells (MEEC) and Mouse Endometrial Stromal Cells (MESC)
5. Vimentin/Cytokeratin Double Staining for Validation of Pure MEEC and MESC Cultures
6. In Vitro Decidualization in a Coculture System
NOTE: Four to five animals are required to establish a coculture system in a 24-well plate. Continue with the following protocol in a sterile environment, preferably under a laminar flow cabinet.
7.Validation of Decidualization by qRT-PCR
NOTE: For validation of the decidualization by qRT-PCR, it is recommended to perform all test-conditions in duplicate in order to receive a sufficient amount of cells for RNA analysis.
Quantitative RT-PCR is performed as described previously in De Clercq et al.8.
In brief:
General Overview of Protocol
Figure 1A illustrates a general overview of the protocol. A daily injection of E2 (100 ng) for three consecutive days was used to synchronize the animals and standardize the phase of the estrous cycle. The estrous cycle can be assessed by vaginal lavages and is divided into the proestrus, estrus, diestrus, and metestrus phase. The cycle phase can be designated according to the ratio of desquamated cells in the lavage, which are subjected to hormonal changes. Increasing levels of estrogen will make the animals evolve to the estrus phase. This phase can easily be detected by the presence of exclusively cornified epithelial cells and the absence of leukocytes (Figure 1B). On day 4, uteri of animals that are in estrus phase are collected and MEEC and MESC are isolated (Figure 1C). Decidualization can be induced by adding 0.5 mM cAMP and 1 µM MPA to the 2% FBS medium during an incubation period of 5 d.
Quality Control of MEEC and MESC Cultures
The purity of the primary cell cultures can be assessed by the difference in vimentin and cytokeratin expression. Vimentin is an intermediate filament that is expressed in mesenchymal cells, hence in the endometrial stromal cells. Cytokeratin is an intermediate filament found in the cytoskeleton of epithelial tissue. Figure 2A shows the mRNA expression level of vimentin and cytokeratin in MEEC and MESC assessed using qRT-PCR. Vimentin levels are high in MESC and low in MEEC (2.2x higher, p < 0.001), while cytokeratin levels are high in MEEC and low in MESC (36x higher, p < 0.001). A vimentin/cytokeratin double staining was performed and indicated that stromal cells express vimentin whereas epithelial cells express cytokeratin (Figure 2B, C). The absence of primary antibody was used as a negative control for the immunostaining (Figure 2D, E). These immunohistological stainings show that both endometrial cell cultures (MEEC and MESC) have a purity of up to 90%.
Decidualization in MEEC/MESC Cocultures
After isolation, mouse endometrial and stromal cells were seeded in a co-culture system to mimic the endometrial environment. Cells were cultured until the epithelial cells formed a monolayer in the cell culture insert (TEER values of 800 – 1,000/well), and the stromal cells reached a sub-confluent state. Decidualization was induced in the stromal cells with the application of 0.5 mM cAMP and 1 µM MPA. After five days of incubation, prolactin mRNA levels were assessed in the stromal cells by qRT-PCR as an indication for decidualization (Figure 3A). Prolactin levels were highly expressed in cells subjected to the hormones and nondetectable in cells incubated with the control medium (p < 0.01).
Since epithelial cells are hard to visualize inside the cell culture inserts, a viability assay was performed immediately after the five-day incubation period (Figure 3B). A mix of normal growth medium and a viability reagent was added to the wells and incubated for a period of minimal 8 – 12 h. The color shift from blue to bright pink indicated the presence of viable epithelial cells. Note that the color shift will only occur when viable cells are present.
Figure 1: General Overview of the Protocol. (A) Scheme of the isolation protocol starting with three consecutive days of estrogen injections. On day 3, the necessary preparations should be prepared. On day 4, estrus phase is evaluated (indicated by star) by performing a vaginal lavage. MESC and MEEC are isolated using different digestion steps. On day 6, or when cells are confluent, decidualization can be induced in the co-culture system by adding cAMP and MPA to the medium and an incubation period of five days (day 6 – 11). (B) Representative picture of the estrus phase characterized by the presence of cornified epithelial cells in the vaginal lavage (magnification 10X). (C) Representative picture of MESC and MEEC (magnification 20X, scale bar: 100 µm). Please click here to view a larger version of this figure.
Figure 2: Immunostaining and Quantitative RT-PCR for Vimentin and Cytokeratin in MEEC and MESC. (A) Messenger RNA levels of vimentin and cytokeratin expression to indicate the purity of the cultures. RNA levels were relatively quantified to the geometric mean of housekeeping genes ACTB and TBP. Data were shown as mean ± SEM. Vimentin/cytokeratin double staining on MESC cultures (B) and MEEC cultures (C). Insert in the left shows a 63X magnification view. Negative control with primary antibody omitted for MESC (D) and MEEC (E) (Scale bar: 50 µm). ***: p <0.001 with Two-way ANOVA with Bonferroni correction for multiple testing. Please click here to view a larger version of this figure.
Figure 3: Validation of Decidualization via RT-PCR. (A) mRNA levels of prolactin expression in stromal cells to evaluate decidualization. RNA levels were relatively quantified to the geometric mean of housekeeping genes PGK1 and TBP. The fold change in cells cultured in control or decidual medium is shown as mean ±SEM. (B) Illustrative pictures of stromal cells before (top) and after (bottom) decidualization stimulus. The insert on the right illustrates a magnification of a decidualized stromal cell. Decidualization is characterized by the presence of multinucleated cells, indicated by black arrows. (C) Representative images of the assessment of epithelial cell viability in the insert after the assay. Pictures were taken immediately after the administration of the viability reagent (Prestoblue) (0 h) and the following morning (ON). **: p <0.01 with Mann-Whitney U test. ON: overnight. Scale bar: 100 µm. Please click here to view a larger version of this figure.
Decidualization is the progesterone-dependent differentiation of endometrial stromal cells into round secreting decidual cells. In human, this process occurs spontaneously during the luteal phase of the menstrual cycle and is initiated in the stromal cells surrounding the vascular cells to form the predecidua. However, in rodents, the presence of a blastocyst is imperative to induce decidualization. The fact that decidualization only occurs after contact with the endometrial epithelial cells implies that important factors are secreted by the epithelial cells to induce decidualization in the underlying stroma. Although this difference in the initiation of the decidualization process should be acknowledged, the fact that human decidualization becomes more robust upon embryo implantation suggests that similar mechanisms could be involved. In vitro cell cultures are often used to study the effect of specific molecules during the process of decidualization. Nevertheless, the availability and amount of human tissue that can be collected is often limited and accompanied by variations, e.g., cycle phase, number of pregnancies and presence of gynecological diseases like endometriosis or adenomyosis. Many of these issues can be prevented by the use of murine tissue that is easily accessible and cycle phase independent. Another strong advantage of using murine tissue is the opportunity to use genetically modified rodents and the possibility to setup a large number of experiments. At the moment, many different isolation protocols have been described in literature with high variability in the age of the animals and the period in the estrus phase at the moment of use.
This study presents a new method for primary endometrial co-cultures of stromal and epithelial cells in which advantage was taken of a standardized estrous cycle by a daily injection of estrogen prior the isolation. Furthermore, estrogen is known to induce proliferation in epithelial and stromal cells which further increases the yield per isolation. The isolation protocol described in this paper was based on Grant et al.7, however, further optimization steps were included to increase the yield, purity, and viability of the different cell cultures. First, HBSS was always supplemented with antibiotics to avoid contamination of the primary culture. During the isolation of MEEC, a temperature adaptation was done (15 min at 37 °C) to increase the harvest of the epithelial cells during the first digestion. Next, an additional step was included to temper enzymatic activity after trypsin-digestion by adding medium containing FBS to inhibit its activity. Furthermore, MEECs were passed through a filter of which the mesh was larger than what is commonly described (100 µm) as they often come in clusters and cell sheets. Moreover, contamination by stromal cells was reduced by performing an additional step in which MEECs and MESCs were separated based on gravity sedimentation. Finally, MEECs were seeded on collagen-coated coverslips to increase the attachment of cells to glass coverslips, as it became clear that attachment to uncoated or PLL-coated glass coverslips was insufficient. During the isolation of the MESCs, collagenase (1 mg/mL) was supplemented to improve digestion. Furthermore, this digestion step was performed in duplicate to avoid contamination of remaining MEECs. All these additional steps resulted in pure and viable cultures of homogenous cell populations.
The purity of the cell population was evaluated with immunostaining and qRT-PCR using vimentin as a stromal marker and cytokeratin as an epithelial marker. Clearly, an opposite expression pattern of vimentin and cytokeratin was observed in MEEC and MESC. However, it can be noticed that the relative mRNA expression of vimentin and cytokeratin is similar in the MEEC cultures. Similar results are published by other research groups, in which epithelial cells in culture acquire vimentin expression9. More important is the difference in protein expression between vimentin and cytokeratin as was observed in the immunohistological stainings of the different cell populations. Double immunostaining showed that EECs were positive for cytokeratin and ESC were positive for vimentin. The use of these markers in immunostaining is an established method and standardly used to indicate the cell types10.
Although a lot of research has been performed on the decidualization of MESC, it remains possible that the presence of epithelial cells in this model may alter the results of decidualization. Firstly, it has been shown that if MEEC grow to a monolayer in the presence of MESC-conditioned medium or in a co-culture setting, the monolayer has an improved epithelial cell transepithelial electrical resistance (TEER), resulting from factors secreted by MESC7. Moreover, Pierro et al.11 provided evidence that epithelial proliferation, influenced by 17β-estradiol, might be mediated by factors secreted from the stromal cells, and alternatively, epithelial cells can release factors that modulate stromal decidualization12,13. Overall, it is clear that the epithelial-stromal co-culture environment provides a more physiological situation that allows for paracrine interaction and communication. The method of culturing two different cell types using an insert co-culture system was described earlier14,15 and was adapted for the use of murine primary cells. By the detection of prolactin mRNA levels as a read-out for decidualization, the described technique has a very reproducible read-out for decidualization available and allows thereby the study of the epithelial-stromal relationship during decidualization. Paracrine signals mediated from MEEC might alter the extent of decidualization in the stromal cells. Moreover, the model allows for specific modulation of the epithelial side without directly affecting the stromal cells. Therefore, this co-culture of MEEC and MESC offers a perfect tool to evaluate the effect of hormones, cytokines, etc. on epithelial cells with a read-out on stromal decidualization. Another advantage of this co-culture system is that it allows researchers to investigate the involvement of a specific protein in the decidualization-process in either the epithelial or stromal compartment by using cells isolated from transgenic animals. Furthermore, this technique will be very helpful to investigate blastocyst adhesion to evaluate whether paracrine factors secreted by epithelial cells in the presence of a blastocyst are sufficient to induce decidualization of the underlying stromal cells. An important step in trophoblast invasion correlated to extracellular matrix degradation, which is mediated by the action of proteolytic enzymes. The proposed technique can be applied as an in vitro model to investigate the cross-talk among the blastocyst, the epithelial cells and the supporting stroma.
However, at the moment little is known about the origin of the epithelial cells which can be as well luminal as glandular. Therefore, further characterization of the genetic and molecular profile of the epithelial cells is required. In addition, the described method is limited in the available knowledge about the polarization of the epithelial cells. At the moment, the polarization of epithelial cells was not taken into account. However, differences in expression of membrane proteins are described in apical and basal membranes. Inappropriate polarization of the epithelial layer could have an impact on the paracrine interaction between epithelial and stromal cells. However, methods to obtain polarized epithelial cells has been described and could be included in the described technique15,16.
Altogether, the described protocol proposes a technique to successfully establish primary cell cultures of mouse endometrial epithelial and stromal cells. This technique results in pure cell cultures as confirmed by qRT-PCR and immunostaining of vimentin and cytokeratin. Ultimately, an epithelial and stromal co-culture was introduced that allows for the investigation of paracrine signals mediated by either MEEC and/or MESC in the decidualization process.
The authors have nothing to disclose.
This work was supported by grants from the Research Foundation-Flanders (FWO G.0856.13N) and the Research Council of the KU Leuven (OT/13/113). K.D.C. is funded by the FWO Belgium. A.H. is funded by OT/13/113. We would like to thank the Cell Imaging Core facility of the KU Leuven (http://gbiomed.kuleuven.be/english/corefacilities/microscopy/cic/cic.htm) for the use of the confocal microscope.
ThinCert Tissue culture insert for 24 well plates; 1 µm pore size; transparant | Greiner Bio-one | 662610 | Inserts are also provided for other multiwell plates and/or other pore sizes. Other suppliers: Merck Millipore. https://shop.gbo.com/en/row/articles/catalogue/article/0110_0110_0090_0030/13408/ |
Nunc Cell culture treated 24 well plates | Thermo Scientific | 142475 | http://www.thermofisher.com/order/catalog/product/142475 |
8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt | Sigma Aldrich | B7880 | Prepare a stock solution of 100 mM in Milli Q water. Store aliquots at -20 °C. http://www.sigmaaldrich.com/catalog/product/sigma/b7880?lang=en®ion=BE |
Medroxyprogesterone 17-acetate | Sigma Aldrich | M1629 | Prepare a stock solution of 250 µM in ethanol. Store aliquots at -20 °C. http://www.sigmaaldrich.com/catalog/product/sigma/m1629?lang=en®ion=BE |
Arachis oil | Supermarket | Standard cooking arachis oil that can be found in every supermarket is used |
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PrestoBlue cell viability reagent | Thermo Scientific | A13261 | https://www.thermofisher.com/order/catalog/product/A13261?ICID=cvc-prestoblue-c1t1 |
HBSS 1x, no calcium, no magnesium, no phenol red | Gibco | 14175-046 | Store in fridge as long as possible. Use cold HBSS+ during the protocol. https://www.thermofisher.com/order/catalog/product/14175046 |
17-β Estradiol | Sigma Aldrich | E8875 | Prepare a stock solution of 1mg/ml in EtOH before diluting in arachis oil (1:1000). http://www.sigmaaldrich.com/catalog/product/sigma/e8875?lang=en®ion=BE |
Cell medium filter Stericup, 0.22 µm, polyethersulfone, 500 mL, radio-sterilized | Merck Millipore | SCGPU05RE | http://www.merckmillipore.com/BE/en/product/Stericup-GP%2C-0.22%C2%A0%C2%B5m%2C-polyethersulfone%2C-500%C2%A0mL%2C-radio-sterilized,MM_NF-SCGPU05RE |
Penicillin-Streptomycin (5,000 U/mL) | Gibco | 15070-063 | https://www.thermofisher.com/order/catalog/product/15070063?ICID=search-15070-063 |
DMEM/F12, phenol red, L-glutamine, HEPES | Gibco | 11330-032 | https://www.thermofisher.com/order/catalog/product/11330057?ICID=search-11330-057 |
Fetal Bovine Serum, qualified, heat inactivated, E.U.-approved, South America Origin | Gibco | 10500-056 | https://www.thermofisher.com/order/catalog/product/10500064?ICID=search-10500-064 |
Amphotericin B | Gibco | 15290-018 | https://www.thermofisher.com/order/catalog/product/15290018?ICID=search-15290-018 |
Gentamicin (50mg/ml) | Gibco | 15750-037 | https://www.thermofisher.com/order/catalog/product/15750037?ICID=search-15750-037 |
DMEM, low glucose, pyruvate, no glutamine, no phenol red | Sigma Aldrich | D5921 | http://www.sigmaaldrich.com/catalog/product/sigma/d5921?lang=en®ion=BE |
MCDB-105 | Sigma Aldrich | M6395 | Dilute in Milli Q water, adjust pH to 7 with NaOH and filter before use. Store in the dark. http://www.sigmaaldrich.com/catalog/product/sigma/m6395?lang=en®ion=BE |
Insulin from bovine pancreas | Sigma Aldrich | I1882 | http://www.sigmaaldrich.com/catalog/product/sigma/i1882?lang=en®ion=BE |
Coverslips, glass, 18 mm Ø | Glaswarenfabrik Karl Hecht | 1001/18 | http://www.hecht-assistent.de/187/?L=1&tx_rmproducts_pi1[g]=937&tx_rmproducts_pi1[p]=3504&tx_rmproducts_pi1[v]=20088&cHash=abd12065683fe74b00eaa5e562824d06 |
Poly-L-lysine | Sigma Aldrich | P2636 | http://www.sigmaaldrich.com/catalog/product/sigma/p2636?lang=en®ion=BE |
Collagenase type IA from Clostridium histolyticum | Sigma Aldrich | C2674 | http://www.sigmaaldrich.com/catalog/product/sigma/c2674?lang=en®ion=BE |
DPBS, no calcium, no magnesium | Gibco | 14190-094 | https://www.thermofisher.com/order/catalog/product/14190094?ICID=search-14190-094 |
Trypsin from bovine pancreas | Sigma Aldrich | T7409 | http://www.sigmaaldrich.com/catalog/product/sigma/t7409?lang=en®ion=BE |
Trypsin-EDTA (0.05%), phenol red | Gibco | 25300-054 | Warm to room temperature before use. https://www.thermofisher.com/order/catalog/product/25300054?ICID=search-25300-054 |
Cell strainer, 40 µm mesh, disposable | BD Falcon | 352340 | https://www.fishersci.com/shop/products/falcon-cell-strainers-4/p-48680 |
Cell strainer, 100 µm mesh, disposable | BD Falcon | 352360 | https://www.fishersci.com/shop/products/falcon-cell-strainers-4/p-48680 |
Millex-GP Syringe Filter Unit, 0.22 µm, polyethersulfone, 33 mm, gamma sterilized | Merck Millipore | SLGP033RS | https://www.merckmillipore.com/BE/en/product/Millex-GP-Syringe-Filter-Unit%2C-0.22%C2%A0%C2%B5m%2C-polyethersulfone%2C-33%C2%A0mm%2C-gamma-sterilized,MM_NF-SLGP033RS?bd=1 |
Acetic acid (glacial) 100% | Merck Millipore | 1.00063 | https://www.merckmillipore.com/BE/en/product/Acetic-acid-%28glacial%29-100%25,MDA_CHEM-100063 |
Collagen I | Corning | 354236 | From rat tail. https://catalog2.corning.com/LifeSciences/en-BR/Shopping/ProductDetails.aspx?categoryname=&productid=354236%28Lifesciences%29# |
Pancreatin from porcine pancreas | Sigma Aldrich | P3292 | http://www.sigmaaldrich.com/catalog/product/sigma/p3292?lang=en®ion=BE |
35 mm Tissue Culture Dishes, Polystyrene, Sterile | BD Falcon | 353001 | https://us.vwr.com/store/catalog/product.jsp?product_id=4675630 |
Ethanol absolute AnalaR NORMAPUR ACS, Reag. Ph. Eur. analytical reagent | VWR Chemicals | 20821296 | https://uk.vwr.com/store/catalog/product.jsp?catalog_number=20821.310DP |
monoclonal rabbit anti-human vimentin (D21H3) | Cell Signalling Tech | #5741 | use 1/500. http://www.cellsignal.com/products/primary-antibodies/vimentin-d21h3-xp-rabbit-mab/5741 |
monoclonal mouse anti-human pan cytokeratin | Sigma Aldrich | C2562 | use 1/1000. http://www.sigmaaldrich.com/catalog/product/sigma/c2562?lang=en®ion=BE |
Paraformaldehyde Solution, 4% in PBS | Affymetrix | 19943 | http://www.affymetrix.com/catalog/130621/USB/Paraformaldehyde+Solution+4+percent+in+PBS#1_1 |
Triton X-100 | Sigma Aldrich | X100 | http://www.sigmaaldrich.com/catalog/product/SIAL/X100?lang=en®ion=BE&gclid=Cj0KEQjwhN-6BRCJsePgxru9iIwBEiQAI8rq879lESeuIU4N4AEQRLNEXj4f8z65KkD-q8Xr35sQDRgaAp-k8P8HAQ |
Goat serum | Sigma Aldrich | G9023 | http://www.sigmaaldrich.com/catalog/product/sigma/g9023?lang=en®ion=BE |
Goat anti-rabbit Alexa Fluor 488 | Thermo Scientific | A-11034 | http://www.thermofisher.com/order/genome-database/searchResults?query=488&resultPage=1&resultsPerPage=15&autocomplete=&searchMode=keyword&productTypeSelect=antibody&targetTypeSelect=antibody_secondary&species=ltechall&keyword=488#filters=genericsort2:%22Capra%20hircus%22;;generic6:^%22Oryctolagus%20cuniculus%22$;;conjugates:^%22Alexa%20Fluor®%20488%22$;; |
Goat anti-mouse Alexa Fluor 594 | Thermo Scientific | A-11032 | http://www.thermofisher.com/order/genome-database/searchResults?query=594&resultPage=1&resultsPerPage=15&autocomplete=&priorSearchTerms=&searchMode=keyword&productTypeSelect=antibody&targetTypeSelect=antibody_secondary&species=ltechall&keyword=594#filters=genericsort2:%22Capra%20hircus%22;;generic6:^%22Mus%20musculus%22$;; |
VECTASHIELD mounting medium with DAPI | Vector Laboratories | H-1200 | http://vectorlabs.com/vectashield-mounting-medium-with-dapi.html |
DPBS, with calcium and magnesium | Gibco | 14040174 | https://www.thermofisher.com/order/catalog/product/14040133 |