A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.
Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.
Cell fate decisions observed in stem cell compartments are tightly regulated by both intrinsic and extrinsic factors. In particular, it is now widely recognized that the BM microenvironment plays a fundamental role in controlling the switch in HSCs from a quiescent to an active state, as well as in their self-renewal or differentiation fate decision1. Moreover, recent findings indicate that hematological malignancies affect the function of the BM microenvironment, pointing to the existence of active crosstalk between the two compartments2,3,4,5,6. Despite recent advances, many key questions remain about how the activity of specific BM-niche components contribute to HSC behavior and malignant transformation.
The BM microenvironment is a highly heterogeneous and complex mixture of many different cell types, each with specialized functions. The abundant endothelial (EC) and vascular component regulate nutrient and metabolite turnover, the ingress and egress of different cells to and from the BM, and several HSC functions7,8. Mesenchymal stromal cells (MSCs), a heterogeneous population of undifferentiated stem cells and progenitors committed through three different lineages (i.e., osteogenic, chondrogenic, an adipogenic), are another fundamental component of the BM niche. These MSCs localize both in central areas of the BM and in the proximity to the endosteal region. They may be associated with vascular structures and are implicated in the regulation of HSC function9,10,11,12,13,14,15.
Many reports suggest that HSCs reside in various defined sites within the marrow and that their function might depend upon their precise localization. Most of the present knowledge regarding HSCs and their interaction with the BM microenvironment derives from murine studies1. The use of xenograft models has extended this knowledge to human normal and malignant HSCs, engrafting within the murine BM of immunodeficient mice16,17,18,19,20. Although this represents a valid model, it still presents many challenges, such as the need to condition the recipient mouse in most cases to allow for human HSC homing and engraftment or the cross-species barrier and its poorly understood influence on cell-cell interactions and functions.
The use of neutralizing antibodies and genetically modified mice, along with xenotransplantation, has been instrumental in highlighting the complex dialog that human HSCs establish with their microenvironments. The introduction and development of intravital two-photon confocal microscopy has moved these studies a step forward, allowing for the direct, high-resolution, and dynamic imaging of the bone marrow19,20,21,22 and provides a powerful tool for the functional characterization of the BM microenvironment and its role in regulating HSC function. In order to circumvent some of the problems arising in classical xenotransplantation models, the concept of engineering a humanized BM structure has been brought to the fore. Merging biomaterials and cell-implantation concepts, reports have shown the feasibility of mimicking the human bone marrow microenvironment in heterotopic regions23,24,25,26,27. This opens the possibility of using bioengineering in mouse models to study human normal and malignant hematopoiesis28,29,30,31,32,33,34,35,36,37,38,39, tumorigenesis, and metastasis40,41,42,43,44.
Based on previous experience in bone tissue engineering and in vivo imaging19,22,45,46,47,48,49,50,51,52, we describe a protocol to bioengineer and live-image organotypic human BM tissues. These structures originate from the implantation of human BM-derived stromal cells into collagen-based scaffolds subcutaneously grafted in immunodeficient mice. In a previous report, we demonstrated that human MSCs ensure the formation of a human microenvironment adequate for the engraftment of human hematopoietic cells45. Furthermore, here we describe how the co-implantation of other human BM cellular components, such as human endothelial cells (hEC), and/or cytokines important for bone formation (e.g., hBMP2), cooperate with hMSCs to generate different humanized microenvironments, which can be live-imaged in situ.
All animal experiments were performed under PPL 70/8904, approved by the U.K. Home Office and in accordance with Cancer Research UK guidelines. The use of human umbilical cord blood (UCB) and primary human acute myeloid leukemia (AML) samples was approved by the East London Ethical Committee after receiving consent and was carried out in accordance with the Declaration of Helsinki.
1. Bioengineering Collagen-based Scaffolds with Human Hematopoietic and Stromal Cells
NOTE: The entire protocol should be performed in sterile conditions and with sterile material. Cell culture medium 1 corresponds to hMSC medium (MEM-α, P/S, and 10% hMSC-FBS); cell culture medium 2 corresponds to EC medium (M199, 20% FBS, P/S, 10mM HEPES, 50 µg/mL heparin, 2 mM glutamine and 50 μg/mL ECGS) and cell culture medium 3 corresponds to hematopoietic cell medium (H5100 and P/S).
2. Surgical Implantation of Bioengineered Scaffolds
NOTE: Here, either male or female, 6- to 12-week-old NSG mice were used. As the animals are immunodeficient, all procedures should be done in sterile conditions. Steps 2.10 – 2.13 are related to survival strategies and post-surgical care.
3. Mouse Treatments, Euthanasia, and Sample Retrieval for Imaging
NOTE: Analysis of the scaffolds is performed between 8 and 24 weeks post-implantation.
4. Live-imaging Using Two-photon Microscopy
NOTE: When using non-descanned detectors (NDD), always use the NDD slider for imaging to direct the fluorescence to the NDD. The microscope configuration is provided in Figure 3.
5. Sample Processing for Histology and Immunostaining
NOTE: Samples are processed according to the protocol described in the JoVE general laboratory techniques54 describing sample fixation, embedding, and sectioning processes. Bone-forming samples should be treated for 7 days in an EDTA-based decalcifying agent between the fixation and embedding processes. The blocking/permeabilization solution is 10 mM PBS pH 7.4 buffer with 1% Triton X-100, 1% bovine serum albumin (BSA), and 10% normal goat serum (NGS).
In Figure 1, representative images of the scaffold cell seeding and implantation processes are shown. In Figure 1C, note that cells are injected directly into the scaffold. In Figure 1G, note that an incision is made in the back of the mouse, where the subcutaneous pocket is created and the scaffold is implanted. Figure 2 shows the gross morphology of different scaffolds implanted in NSG mice and retrieved after 8 weeks. Note the slight vascularization in hMSC seeded scaffolds (Figure 2A). The co-seeding of human ECs with hMSCs in the scaffold allows for the formation of more relevant vasculature in scaffolds (Figure 2B). Finally, the presence of rhBMP-2 induces bone formation. The retrieved scaffolds are bigger in this case, and they are constituted by bone-resembling hard tissue.
Figure 3 shows the channel configuration setup on the microscope for live-imaging with NDD (details in the figure legend). Figure 4 and Video 1 show human hematopoietic cells in hMSC-coated scaffolds. Scaffolds were explanted 8 weeks post-implantation and after the intravenous inoculation of AF488-hCD45 antibody and 655-VPA. This procedure allows for the visualization of implanted human hematopoietic cells and the vascular structure by two-photon confocal microscopy. In this case, the images show blood vessels (655-VPA) in scaffolds and the long-term engraftment of human hematopoietic cells (AF488-hCD45) in the scaffold parenchyma. Figure 5 and Video 2 correspond to human scaffolds seeded with hECs and hMSCs. 8 weeks after surgery, scaffolds were explanted after the intravenous inoculation of FITC-hCD31 antibody and 655-VPA, and images were acquired with a two-photon confocal microscope, as mentioned before. Images show the participation of hECs in vessel formation in the scaffold, resulting in a murine-human chimeric vasculature.
Figure 6A shows representative data of the approach used to stimulate bone formation in MSC scaffolds. Similar to previous figures, 8 weeks after implantation, the intravenous inoculation of 655-VPA was performed, scaffolds were retrieved, and images were acquired with two-photon confocal microscopy. rhBMP-2-stimulated scaffolds induce the formation of bone tissue, which could be visualized due to the SHG (cyan color in the images) provided by the calcium in the bone. The provided images also show the formation of cavities and vascularized endosteal tissue, which highly resemble the BM endosteal tissue. In Figure 6B and Video 3, hECs were co-implanted with hMSCs. Scaffolds were retrieved after the intravenous inoculation of FITC-hCD31 antibody and 655-VPA, and two-photon confocal microscopy images show the participation of hECs in the neovascularization in a bone-forming scaffold.
Figure 7 shows representative images of histology, a procedure performed to corroborate previously described results. Immunofluorescent images show mouse vasculature, hECs, hMSCs, and long-term engrafted human hematopoietic cells in the scaffold structures. Scaffolds retrieved from mice were fixed and used for immunofluorescence. In the rhBMP-2 carrier bone-forming scaffolds (Figure 7D-F), note the morphology of the tissue, resembling mature bone marrow with adipose tissue. In this bone-forming scaffold, we show that hMSCs are fibroblasts, which would indicate that they contribute to newly formed tissue as stromal cells. We also show human adipocyte marker expression, which would indicate that hMSCs also contribute to adipose tissue formation.
Figure 1. Representative Images of the Cell-seeding and Implantation Processes. A) Initial scaffold and its cutting method using a scalpel. B) 24 pieces obtained from the initial scaffold. C) Scaffold cell-seeding method using a syringe. D) Cell-seeded scaffolds with culture medium, ready to be implanted. E-F) Specific steps for bone-forming scaffolds: E) scaffold being transferred to a 96-well, u-bottom plate and F) representative image of the method used to add rhBMP2, thrombin, and fibrinogen to the scaffold. G-J) Surgical implantation procedure under general anesthesia: G) wound created in the skin and scaffold implantation, H) implantation method, and I-J) wound closing procedure using surgical glue. Please click here to view a larger version of this figure.
Figure 2. Different Scaffolds Retrieved from Mice. Representative images of MSC scaffolds (left), MSC+EC scaffolds (middle), and MSC+EC+BMP scaffolds (right). Please click here to view a larger version of this figure.
Figure 3. Channel Configuration. The microscope filter setup is shown. A) There are four NDD detector modules: in the first module, there are two filter cubes; the second and third modules have one filter cube each; and the last module has no cube (not used). The first photomultiplier tube (PMT) is for the far-red channel (640 – 690 nm), reflecting the lower wavelengths; the following ones are 380 – 485 nm, 500 – 550 nm, and 555 – 625 nm (the order is always from the lower to higher wavelengths). B) The fluorophore emissions detectable with the above configurations (color coded). Please click here to view a larger version of this figure.
Figure 4. MSC Scaffolds Allow for the Formation of a Niche for Human Hematopoietic Cells. A) and B) 3D reconstructions of Z-stacks taken post-explant, after intravenous inoculation with AF488-hCD45 (green), to label human hematopoietic cells, and 655-VPA (magenta)m to label vasculature. Scale bars represent 20 µm in A and B (upper panels) and 5 µm in B (lower panels). Please click here to view a larger version of this figure.
Video 1. MSC Scaffolds Allow for the Formation of a Niche for Human Hematopoietic Cells. 3D reconstruction of human hematopoietic cells (AF488-hCD45) associated with the vasculature (655-VPA) in the MSC scaffold (collagen structures: SHG, cyan). Each stack measures 140 x 140 µm. Please click here to view this video. (Right-click to download.)
Figure 5. Human ECs Participate in the Formation of Humanized Vessels in MSC Scaffolds. 3D reconstruction of vasculature (655-VPA) lined by ECs of human origin (FITC-hCD31) in MSC+EC scaffolds. Scale bars represent 20 µm. Please click here to view a larger version of this figure.
Video 2. Human ECs Participate in the Formation of Humanized Vessels in MSC Scaffolds. 3D reconstruction of human ECs (FITC-hCD31) lining the vasculature (655-VPA) in MSC+EC scaffolds. Each stack measures 240 x 240 µm. Please click here to view this video. (Right-click to download.)
Figure 6. rhBMP-2 Carrier Scaffolds have Bone Surfaces and Humanized Vasculature Similar to the Bone Marrow. A) 3D reconstruction of MSC+BMP scaffolds showing the formation of bone structures (SHG-cyan) and vasculature (655-VPA). Scale bars represent 100 µm (left), 70 µm (middle), and 50 µm (right). B) 3D reconstruction of MSC+EC+BMP scaffolds showing humanized vessels (655-VPA) lined with human ECs (FITC-hCD31). Scale bars represent 50 µm (left) and 30 µm (middle and right). Please click here to view a larger version of this figure.
Video 3. rhBMP-2 Carrier Scaffolds have Bone Surfaces and Humanized Vasculature Similar to the Bone Marrow. 3D reconstruction of MSC+VERA+BMP scaffold (bone: SHG; vessels: 655-VPA; human ECs: FITC-hCD31). Each stack measures 600 x 600 µm. Please click here to view this video. (Right-click to download.)
Figure 7. Representative Images of Immunofluorescence Performed on Fixed Scaffolds. A-F) Immunofluorescence studies performed to locate human cells implanted in scaffolds. A-C) Scaffolds implanted with hECs, hMSCs, and hHSCs. D-F) Bone-forming scaffolds implanted with hECs, hMSCs, and hHSCs. The color channels are as follows: A-D) human EC (hCD31) and mouse vascular structure (endomucin, ENDOM), B-E) hMSCs (hVimentin, hVIM) and mouse vascular cells (endomucin, ENDOM), C) human hematopoietic cells (hCD45) and mouse vasculature (endomucin, ENDOM), and F) human adipose differentiation-related protein (hADRP) and mouse vasculature (endomucin, ENDOM). Scale bars represent 10 µm (A and C), 20 µm (B and E), and 40 (D and F) µm. Please click here to view a larger version of this figure.
Significance with Respect to Existing Methods:
In this protocol, we described a method to generate different humanized microenvironments in mice and to visualize their architecture via two-photon microscopy and histology. The representative data provided shows the feasibility of the approach, using different stromal cells to engineer humanized tissues. The protocol has specific applications to the study of human hematopoietic cells and bone marrow niche-derived cells in normal and pathological conditions. These applications include the study of clonal evolution, drug screening, and crosstalk between human HSCs and stromal components. In the emerging field of tissue engineering, several alternative approaches have been proposed. Approaches of note include the development of 3D humanized BM structures in vitro55,56,57,58,59,60,61,62,63 and the orthotopic graft of humanized BM scaffolds in mice64. Our approach has the advantage of combining both the complexity of the in vivo system with the easy anatomical accessibility of the humanized tissue graft.
Modifications and Troubleshooting:
A source of variability in this protocol can be found in the selection of cells used to seed the scaffolds. In our work, we used BM-derived hMSCs. However, mesenchymal cells can be obtained from several tissues, which may show distinctive properties depending upon the origin. Therefore, the use of hMSCs derived from different organs can be considered. However, their ability to form bone tissue in vivo should be tested prior to use in this protocol.This protocol uses a commercially available human endothelial cell source (i.e., E4ORF1-transduced HUVEC). Recently, the use of organ-specific endothelial cells for different purposes has been reported65,66. Furthermore, the use of primary hECs derived from the BM could represent an interesting improvement to the protocol. Therefore, the use of different sources of endothelial cells may produce different in vivo outcomes.
We used NSG immunocompromised recipient mice to favor the implantation of humanized scaffolds and to avoid tissue rejection. We do not exclude the possibility of using this protocol to engineer ectopic bone marrow tissues in other mouse strains. Indeed, rhBMP-2 can induce bone formation in different mammalian models47,48,49,50,52. However, differences in cell viability and long-term transplantation are likely to be observed using different strains/models.
The timing of scaffold recovery can also be flexible, depending upon the final purpose of the experiment. In the presented protocol, we recover samples at 8 – 12 weeks after implantation to assess long-term hematopoietic engraftment. To study early steps of the human BM niche formation (e.g., osteochondral tissue formation47 or vascular development), different time points can be chosen.
The live-imaging technique we described in this protocol is indicated for short-term imaging of explants. The use of an equilibrated chamber for maintaining physiological temperature, oxygen tension, and CO2 concentration should be considered in cases of long-term imaging, such as to study motility behaviors.
Critical Steps within the Protocol:
Among the challenges related to the protocol, we would highlight the technical skills required for some steps. Mesenchymal and endothelial cells should be used at low cell passage numbers; otherwise, they will not be able to support human hematopoietic cell engraftment in vivo or to participate in de novo vasculature and bone formation in vivo. We recommend the use of hMSCs and hECs at passages 1 – 5. Scaffold preparation and cell-seeding steps require basic cell-culture skills and knowledge of the properties of the specific cells used in the procedure. The surgery protocol is quite straightforward but requires some practice. Maintenance of an aseptic environment to avoid contamination of the implanted scaffolds in immunodeficient mice is crucial to ensure the success of the experiment. Sample explant and live imaging require surgical practice (especially for the use of the microdrill) and knowledge of the microscope system. Finally, sample processing and histology require basic knowledge of the techniques to be used.
Limitations of the Technique:
The approach we describe allows for the visualization of live human hematopoietic cells seeding a humanized bone marrow microenvironment, with human endothelial cells forming vascular structures and mesenchymal cells forming bone/bone marrow space. As the tissue is formed in vivo, the final engineered scaffold will still be chimeric (human and murine). This issue should be taken into account, as the chimeric tissue may not fully mimic human bone marrow complexity and environment.
The scaffolds implanted have a limited size (we tried a maximum of 6.6 x 7.5 x 7 mm), and therefore, they are able to host a limited number of cells for xenotransplantation. The absolute number of recovered cells will also be limited; thus, the number of implanted scaffolds should be calculated as a function of the number of cells required for the experiment.
The imaging application we described is particularly useful for observing large areas of live tissue at depths of 150-200 µm from the surface without disrupting the architecture and damaging the cells. Therefore, it does not allow for the visualization of the whole scaffold. If a complete scan of the tissue is required, standard immunofluorescence approaches would be more appropriate.
Future Applications:
The future direction of this bioengineered model would be to increase the complexity of the human components in the tissue. The knowledge and characterization of the human BM niche has progressed in recent years67, and the described protocol could be an interesting platform to study the function of these new cellular components and soluble factors, as well as their role in supporting normal/malignant HSCs.
Furthermore, the imaging technique provides the potential for intravital imaging of the scaffolds in longitudinal studies, which would require technical improvements in imaging the scaffolds in live, anesthetized mice, with post-surgery recovery. This approach would require additional steps and is currently under investigation in the laboratory.
The authors have nothing to disclose.
We thank the staff in the core facilities at the Francis Crick Institute (Biological Research facility, In Vivo Imaging and Experimental Histopathology) and Yolanda Saavedra-Torres and Mercedes Sanchez-Garzon, the vets at Crick and LRI respectively, for their valuable help. We are grateful to Dr. W. Grey for critically reading the manuscript. D.P. was supported by a non-clinical research fellowship from the EHA. This work was supported by The Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001045), the UK Medical Research Council (FC001045), and the Welcome Trust (FC001045).
Ficoll-Paque | Ge Healthcare | 17-1440-03 | |
Cd34 positive selection Kit | Stemcell | 18056 | Store a 4°C. |
Magnet | Stemcell | 18000 | |
Anti-human CD3 antibody, clone OKT-3 | Bioxcell | BE0001-2 | Store at 4°C. Used for T cell depletion in primary AML samples. |
Cytokines (IL3, G-CSF, and TPO) | PeproTech | 200-03, 300-23 and 300-18 | For doing the stock: Dilute each one of the cytokines in 100μL of water and mix them. Add 200μL, do alicuots of 45μL and Store at -20°C. |
DMSO | Sigma | D4540 | |
FBS | Life technologies | 10270106 | Heat-inactivate at 56°C during 30 minutes, do aliquots and freeze down. Warm in 37°C water bath before use. |
MEM-α | Invitrogen | 32571-028 | Store at 4°C. Warm in 37°C water bath before use. |
Myelocult H5100 | corning | 5100 | Store at 4°C. Warm in 37°C water bath before use. |
199 | Gibco | 41150-020 | Store at 4°C. Warm in 37°C water bath before use. |
hMSC-FBS, Heat-inactivated | Gibco | 12662-029 | Store at -20°C. Warm in 37°C water bath before use. |
P/S | Sigma-Aldrich | P0781 | Store at -20°C. Warm in 37°C water bath before use. |
ECGS | Millipore | 02-102 | Dilute in culture media and use 0.22 mm filter. |
HEPES | Sigma-Aldrich | S1558-50ML | Store at 4°C. |
Heparin | Sigma-Aldrich | H3149 | Store at 4°C. |
Glutamine | Gibco | 25030 | Store at -20°C. |
Collagen 1 coated cell culture plate | corning | 354505 | |
Trypsin-EDTA solution | Thermo-Fisher | 25200056 | Store at -20°C. Warm in 37°C water bath before use. |
hMSC | Lonza | PT-2501 | Alternatively, hMSCs we also kindly provided by Dr. Dosquet (University Paris Diderot, Paris) from human bone marrow obtained during orthopaedic surgery under ethical approval 10-038 from IRB00006477. |
VeraVec HUVEC endothelial cells | Angiocrine bioscience | hVera101 | |
Human hematopoietic cells | Umbilical Cord blood or primary Acute Myeloid Leukemia (AML) samples were obtained from the Royal London Hospital (London, UK) after informed consent and protocol of use was approved by the East London Ethical Committee and carried out in accordance with the Declaration of Helsinki. | ||
Gelfoam, Size 12-7mm | Pfizer | 00009-0315-08 | |
PBS | Thermo-Fisher | 10010023 | |
Surgical material | Multiple | Sterile forceps, tweezers and sharp scissors. | |
Sterile tissues | Heat-sterilize paper tissues. | ||
1 ml Syringe with needle of 25G | Terumo | SS+01H25161 | |
Ultra Low Attachment Multiple Well Plates | Corning | 3473 or 3471 | |
BMP2 | Noricum | rhBMP-2 | Dilute at 5 mg/ml in acetic acid 50 mM and store at 4 °C. |
Thrombin | Sigma | T9010 | Dilute in CaCl2 2%, 500 mL per vial, and store at 4 °C. |
Fibrinogen | Sigma | F3879 | Dilute at 4 mg/100 mL in PBS. Store at -20C. Warm before use. |
Tissue-culture dishes 35 mm x 10 mm | Falcon | 353001 | |
U-Botton 96 well plate | Falcon | 353077 | |
NSG mice | The Jackson Laboratory | 5557 | NSG mice were a kind gift from Dr Leonard Shultz (The Jackson Laboratory). |
Chlorhexidine | G9 | Dilute 1:10 before use | |
Carprofen | Pfizer | Rimadyl | 5 mg/g of mouse |
Buprenorphine | Alstoe | Vetergesic | 0.1 mg/g of mouse body weight |
Isoflurane | Abbott | B506 | Induction of anaesthesia 2%, maintenance 1% |
Trimmer | Wella | Contura HS61 | |
Surgical glue | 3M | Vetbond | |
carbomer (polyacrylic acid) as Ophthalmic gel | Novartis | Viscotears Liquid Gel | |
Human Normal Immunoglobulin | Gammaplex | 10g vial | 100 ml/mouse intravenously, 30 min before infusion of specific antibody. |
NT-QTracker | Invitrogen | Q21021MP | Vessel-pooling agent. Administrate 15 ml/mouse intravenously 5 min before imaging. |
AF488-hCD45 | Biolegend | 304017 | 100 ml/mouse intravenously, 30 min before imaging. |
FITC-hCD31 | BD Pharmigen | 555445 | 100 ml/mouse intravenously, 30 min before imaging. |
Super glue | Loctite | Super Glue | |
Micro-Drill Kit | IDEAL – Fisher Scientific | NC9010016 | |
Microsurgical microscope | No specific brand/company is adviced. | ||
LSM 710 NLO | Zeiss | Upright confocal microscope with motorized stage, two-photon laser and 20x 1.0 NA water immersion lens. Alternatively, a microscope with similar specifications could be used. | |
MaiTai “High Performance” fully automated 1-box 517 mode-locked Ti:Sapphire laser with DeepSee dispersion compensation | Spectra-Physics | ||
Formalin solution, neutral buffered, 10% | Sigma-Aldrich | HT501128 | |
Ethanol | Sigma-Aldrich | 32294 | |
Osteosoft | Millipore | 1.01728.1000 | |
Polysine slices | Thermo scientific | J2800AMNZ | |
Antigen unmasking solution | Vector | H-3300 | Store at 4 °C. Dilute 1:100 in H2O for working solution. |
Triton 100x | Sigma-Aldrich | T9284 | |
Bovine serum albumin (BSA) | Sigma-Aldrich | A96470 | Store at 4 °C. |
Normal Goat serum (NGS) | Sigma-Aldrich | G9023 | Store at 4 °C. |
Endomucin Antibody | Santa Cruz | sc-65495 | Store at 4 °C. |
hVimentin antibody | Santa Cruz | sc-6260 | Store at 4 °C. |
hCD31 antibody | DAKO | M0823 | Store at 4 °C. |
hCD45 antibody | DAKO | M0701 | Store at 4 °C. |
ADRP (Perilipin2) | antibodies-online | ABIN283918 | Store at 4 °C. |
Goat anti mouse secondary antibodies | Invitrogen | A11029 or A11005 | Store at 4 °C. |
Goat anti Rabbit secondary antibodies | Invitrogen | A11037 or A11008 | Store at 4 °C. |
Goat anti Rat secondary antibodies | Invitrogen | A11007 or A21247 | Store at 4 °C. |
Sudan Black | Sigma | S2380 | Prepare a stock of 1% sudan black in ethanol 70%. Store at RT. Prepare working solution of 0.1% sudan black in ethanol 70% and filter using filter-paper before use. |
DAPI | Sigma | D8417 | Prepare stock in H20 at 100 mg/mg. Store at 4 °C. |
Fluorescent mounting media | Dako | S3023 | Add DAPI before use (1:400 from DAPI stock). |
Cover glass | VWR | 631-0147 |