Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.
Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.
DR is a serious ocular complication of diabetes, a disease that has reached enormous proportions in the last three decades1. Twenty years after diagnosis, virtually every patient with type 1 diabetes and 60% of patients with type 2 diabetes present signs of retinopathy, making diabetes per se one of the leading causes of blindness in working age adults2. According to the level of microvascular degeneration and ischemic damage, DR is classified into non-proliferative DR (non-PDR) and proliferative DR (PDR). The end-stage disease, PDR, is characterized by ischemia- and inflammation-induced neovascularization and fibrotic responses at the vitreoretinal interface. In untreated conditions, these processes will lead to blindness due to vitreous hemorrhage, retinal fibrosis, tractional retinal detachment, and neovascular glaucoma3,4. Despite recent advances, current treatment options target only DR stages, including diabetic macular edema and PDR, when retinal damage has already ensued. Moreover, a great proportion of DR patients does not benefit from current treatment armamentarium, indicating an urgent need for improved therapies4,5,6.
Multiple other in vivo disease/developmental models and diabetic animal models have been developed to date, but none of them recapitulates the full range of pathologic features observed in human PDR7,8. Moreover, increasing evidence indicates that treatment responses are tightly connected to the ECM composition as well as the spatial arrangement and interaction between the cellular and acellular microenvironment9. We, therefore, set out to develop a clinically relevant model of human PDR by utilizing the FT pathological material that is commonly excised from eyes undergoing vitrectomy as part of the surgical management of PDR10.
This manuscript describes the protocol for the 3D ex vivo culture and characterization of the surgically-excised, PDR patient-derived pathological FT. The method described here has been used in a recent publication that demonstrated successful deconstruction of the native 3D PDR tissue landscape, and recapitulation of features of PDR pathophysiology including angiogenic and fibrotic responses of the abnormal vascular structures11. This model also revealed novel features that cannot be easily appreciated from thin histological sections, such as spatially confined apoptosis and proliferation as well as vascular islet formation11. Vitreous fluid has been successfully used by others on 3D endothelial spheroid cultures to evaluate its angiogenic potential and the efficacy of angiostatic molecules12. When combined with an in vitro 3D lymphatic endothelial cell (LEC) spheroid sprouting assay using PDR vitreous as stimulant, our model revealed the contribution of both soluble vitreal factors as well as local cues within the neovascular tissue to the as yet poorly understood LEC involvement in PDR pathophysiology3,11. In the management of PDR, vitreoretinal surgery is a routinely performed yet challenging procedure. As surgical instrumentations and techniques are seeing continuous advancement and sophistication, timely and conservative removal of fibrovascular proliferative specimen not only improves vision outcome but also provides invaluable tissue material for the investigation of PDR pathophysiology and treatment responses in the complex translational aspects of the live human tissue microenvironment.
This research was approved by the Institutional Review Board and Ethical committee of Helsinki University Hospital. Signed informed consent was obtained from each patient.
1. Preparation of Solutions, Media and Equipment
2. Fibrovascular Tissue Dissection
3. Casting Upright Fibrin Gel Droplets for the Characterization of Native FT and Ex Vivo Culture
4. “In Matrix” Imaging
5. Native FT and Ex Vivo Culture End-Point
NOTE: The FT/fibrin gels can be cultured ex vivo for the desired time period (FT ex vivo cultures) or fixed on the same day (native FT) for native FT characterization11.
6. Whole-Mount Immunofluorescence Staining
NOTE: This protocol lasts 5 days and can be interrupted with overnight incubations where indicated. Do not let the fibrin gels dry at any point. All steps are performed at RT unless otherwise indicated.
Deeper understanding of the PDR fibrovascular tissue properties and protein expression has relied mainly on vitreous samples and thin histological FT sections3,15,16,17. To develop a method for thorough investigation of the 3D tissue organization and multicellular physiopathological processes of PDR, we set out to utilize the surgically excised, patient-derived pathological FTs for 3D characterization and ex vivo culture. The fresh FTs are transferred to the research laboratory and processed as illustrated in the schematic workflow in Figure 1.
The FTs can be imaged prior to dissection for qualitative assessment and documentation (Figure 2). As shown in Figure 2, the FTs display great inter-patient variation in size, density and abundance of vascular structures. In some tissues, loose cells, presumably inflammatory/immune cells or red blood cells, as well as pervious vascular structures of different caliber can also be easily distinguished (Figure 2). After dissection, a decision needs to be made on the downstream usage of the limiting number of the obtained explants. A portion of the explants is embedded within fibrin matrix and fixed after fibrin gel formation while the remaining explants can be subjected to ex vivo culture on a separate plate (Figure 1). The fresh FTs have also been successfully utilized for ultrastructural characterization by electron microscopy. The samples can be either processed for conventional transmission electron microscopy (TEM) of ultrathin sections or for serial block face-scanning electron microscopy (SBF-SEM)3,11.
The fixed fibrin gels can be stored for several weeks and stained by whole-mount immunofluorescence18,19. The stained samples are likewise stable when stored at 4 °C in the dark. The thick FT/fibrin gels can be imaged time-efficiently with epifluorescence microscope equipped with an optical sectioning function, useful for removing scattered out-of-focus light. Imaging with this method provides fine visualization of the structures. Alternatively, confocal microscopy, though more time-demanding, can allow the capture of finer details. The characterization of the freshly fibrin-embedded and fixed, uncultured, native FTs by whole-mount immunofluorescence allows the characterization of vascular structures, multiple cell types and their reciprocal arrangement within the 3D PDR tissue landscape11. For example, CD31 antibodies can be used to display the endothelium and NG2 to display pericytes (Figure 3) while Lyve1 antibodies visualize newly discovered lymphatic-like endothelial structures (Figure 4)11. Multiple combinations of antibodies can be used to visualize several structures and cell types (see Table of Materials); for example, ERG can also visualize the endothelium, which has a discontinuos expression pattern in the abnormal PDR neovasculature. The vascular structures stained with a membrane marker (e.g., CD31 and Lyve1) can be quantitatively analysed for preservation and density by using Angiotool, a free-source software developed by the NIH National Cancer Institute11,20. 3D volumes can also be rendered from the dataset obtained (see Video 1). If an antibody is not suitable for whole-mount immunofluorescence, the fibrin droplets can alternatively be embedded in paraffin, cut to thin sections and analyzed by immunohistochemistry. In this case the droplets benefit from overnight fixation at 4 °C instead of the 1h at RT.
Ex vivo culture sustains the growth of the fibrin-embedded FTs, developing cellular outgrowths already after two days (Figure 5). The in vitro fibrin gel is used as the matrix for ex vivo culture, since it typifies the in vivo provisional ECM, formed after thrombin cleavage of serum fibrinogen at the sites of vascular leakage, in direct contact with the leaky PDR neovessels in the inflamed fibrotic milieu15,21,22.
PDR is a microvascular complication characterized by an angiogenic and fibrotic response, and the FT explants retain this cellular repertoire in culture, as well as respond efficiently to exogenous stimuli added to the culture media11. The representative data in Figure 6 shows that the PDR ex vivo cultures induced sprouting of the CD31-positive endothelium and vasculature preservation in response to VEGFA, while TGFβ induced a fibrotic response reflected by the outgrowth on NG2-positive pericytes/SMCs. Several exogenous stimuli can be tested and, when informed by measurements of their in vivo vitreal abundance, the herein developed PDR ex vivo culture model can reveal novel microenvironment and context dependent PDR pathological mechanisms that cannot be visualized in fixed tissue material.
Figure 1. Ex vivo PDR fibrovascular tissue culture model. Schematic representation of the workflow from the vitreoretinal surgery (pars plana vitrectomy) for the excision of the FT to its dissection into explants and embedding into fibrin for three-dimensional characterization and ex vivo culture. Please click here to view a larger version of this figure.
Figure 2. Freshly excised PDR fibrovascular tissues prior to dissection. Phase contast micrographs of freshly excised FTs taken prior to dissection. The FTs are highly variable in size, density and abundance of vascular structures. Arrowheads indicate vascular structures of different caliber. The red partially transparent line highlights two individual vessels of different caliber. The images were taken using an inverted epifluorescence microscope with a 5x, 0.15 numerical aperture (NA), objective. Please click here to view a larger version of this figure.
Figure 3. Whole-mount immunofluorescence of a native PDR fibrovascular tissue. Epifluorescence micrograph of a native PDR FT stained by whole-mount immunofluorescence. CD31 (A, green) visualizes the endothelium while NG2 (B, red) visualizes the pericytes within the irregular neovascular structures. Merged images are shown in (C). DAPI (blue) counterstain visualizes nuclei. The image was taken using an upright epifluorescence microscope with optical sectioning function and combined with a computer-controlled 1.3 megapixel monochrome CCD camera and image acquisition software, using a 40x, 1.4 NA, oil objective. Nine optical sections were combined by using image processing software ImageJ. Please click here to view a larger version of this figure.
Figure 4. Whole-mount immunofluorescence of a native PDR fibrovascular tissue. Epifluorescence micrograph of a native PDR FT stained by whole-mount immunofluorescence. CD31 (A, green) visualizes the endothelium while Lyve1 (B, red) visualizes the newly discovered lymphatic-like endothelial structures. Merged images are shown in (C). Hoechst-33342 (blue) counterstain visualizes nuclei. The image was taken using an upright epifluorescence microscope with optical sectioning function and combined with a computer-controlled 1.3 megapixel monochrome CCD camera and image acquisition software, using a 20x, 0.8 NA, objective. Four optical sections were combined by using image processing software ImageJ. Please click here to view a larger version of this figure.
Figure 5. Ex vivo growth of the PDR fibrovascular tissues. Phase contrast micrographs of two FT ex vivo cultures at indicated time points (day). The FTs grow upon ex vivo culture, developing cellular outgrowths already after two days. The images were taken using an inverted epifluorescence microscope with a 5x, 0.15 NA, objective. Please click here to view a larger version of this figure.
Figure 6. Whole-mount immunofluorescence of PDR fibrovascular tissues cultured ex vivo untreated (CTRL), or in presence of VEGFA or TGFβ. Epifluorescence micrograph of FT cultured ex vivo untreated (CTRL) or in presence of VEGFA or TGFβ for 9 days and subsequently stained by whole-mount immunofluorescence. CD31 (green) visualizes the endothelium while NG2 (red) visualizes the pericytes. DAPI (blue) counterstain visualizes nuclei. VEGFA stabilized the vasculature while TGFβ induced a fibrotic response. The images were taken using an upright epifluorescence microscope with optical sectioning function and combined with a computer-controlled 1.3 megapixel monochrome CCD camera and image acquisition software, using a 20x, 0.8 NA, objective. Nine optical sections were combined by using image processing software ImageJ. Please click here to view a larger version of this figure.
Video 1. 3D volume reconstruction of a native FT stained by whole-mount immunofluorescence. CD31 (green), Lyve1 (red). Hoechst-33342 counterstain (blue) visualizes nuclei. The image was taken using an upright epifluorescence microscope with optical sectioning function and combined with a computer-controlled 1.3 megapixel monochrome CCD camera and image acquisition software, using a 20x, 0.8 NA, objective. 3D volume reconstruction and video rendering was performed using a commercial software. A portion of the video is reprinted with permission from Gucciardo et al.11. Please click here to view this video. (Right-click to download.)
Considering the importance of relevant tissue microenvironment for reliable functional cell and molecular mechanistic results, it is imperative to find appropriate experimental models that provide this tissue environment. The herein described ex vivo PDR culture model for the fibrin-embedded FTs allows the investigation of the mechanisms of PDR pathophysiology in the native, complex and multicellular context of the PDR clinical samples.
Critical steps within the protocol are the proper fibrin gel formation, the positioning of the FT and adequate washing during the staining. Since fibrin gel formation depends mostly on the thrombin activity, affected by the concentration and temperature, the amount of thrombin needs to be adjusted for every batch of fibrin gel preparation. Fibrin gel formation should be quick enough to prevent 2D growth of the FT explants but also slow enough to allow positioning of the FT to the center of the droplets vertically and horizontally. In the case the FT happens to locate at the edge of the droplet, additional pipetting could aid the placement of the FT to the center. FTs that still remain at the edge of the droplets or that grow in 2D will need to be excluded. Adequate washing during staining and avoiding drying of the droplets are in turn critical in order to optimize staining and minimize background signal. Transfer times may also be critical. We have embedded the FTs up to two hours after vitrectomy and this did not affect the ex vivo growth. The impact of longer transfer times on the culture success would need to be tested.
This protocol could be modified by using alternative matrices for FT embedding. In vivo, the PDR neovessels grow towards the vitreous cortex rich in collagen23. However, upon vascular leakage serum components, including fibrinogen, dissolve into vitreous in close proximity to the vessels whereby the fibrotic response is initiated. Therefore, the in vitro fibrin clot was utilized here for the ex vivo culture in order to typify the provisional ECM formed in the inflamed fibrotic milieu of PDR15,21,22. Alternatively, the fibrin clots could be supplemented with laminin and fibronectin to alter the stability of sprouting capillaries, or the explants could be embedded within type I collagen and other mixed matrices to typify the most fibrotic microenvironments in neovascular tissues. These matrices are generally suitable for 3D culture and whole-mount immunofluorescence but their effects on the PDR FTs remain to be tested and considerations on the timing of 3D matrix formation will need to be made in order to stay within the limits for preventing 2D growth18,19. When the physical proximity of the research unit to the hospital represents a limitation, longer transfer times could be compensated with team work; TA solution preparation, fibrinogen sterile-filtration and fibrin gel formation testing can be performed during FT transfer. If the fibrin gels do not form even after 1 hour, a problem with the fibrinogen or TA solution preparation might have occurred. In this case, the FT/fibrin gels cannot be used.
Limitations of this approach, as with every ex vivo approach, are the variable quality of primary tissue specimen across individuals as well as intra-patient and inter-patient tissue heterogeneity. In the case of diabetic patients, part of this variability includes the extent of vascularization and fibrosis which depend on numerous factors like duration of the disease, metabolic condition, genetic/epigenetic factors, type of diabetes and systemic therapy. The size of the surgical PDR FT recovered is also a limiting factor in determining the number of conditions that can be investigated for each specimen. While providing reciprocal spatial information, whole-mount immunofluorescence allows the investigation of only a limited amount of features/markers per droplet. As a compromise, the fibrin droplets can alternatively be embedded in paraffin, cut to thin sections and analysed by immunohistochemistry.
Existing diabetic mouse models develop many features of early stage DR but fail to comprehensively recapitulate the progressive changes occurring in human PDR, thus hindering the studies of the PDR disease mechanisms7,8. Moreover, the murine eye is fundamentally different from the human eye, in that it lacks the macula, further emphasizing the importance of studying the human disease24. The surgical PDR FTs have previously either been discarded, or used for paraffin sections, transmission electron microscopy, serial block face-scanning electron microscopy, bulk RNA sequencing or 2D culture of dissociated cells3,25. The herein described model allows the 3D characterization of this precious surgical material, as well as the investigation of PDR pathophysiology in the native diseased tissue microenvironment. When combined with the use of vitreous fluid, this model also allows the investigation of the contribution of both the cellular and acellular PDR microenvironment. Since the cultures respond efficiently to growth factors detected in the vitreous, such as VEGFA, bFGF, VEGFC and TGFβ, thus recapitulating features of PDR pathophysiology, this PDR ex vivo culture model is amenable for testing or developing new PDR treatments11. In this model, anti-VEGFA prevented capillary sprouting and induced signs of capillary regression, responses that are consistent with the expected clinical outcomes of anti-VEGFA treatment26,27. Therefore, this model can also be used for better understanding the effects of current therapies, including anti-VEGFA and corticosteroid treatments.
With live-cell imaging instrumentation, the herein described ex vivo culture model could be subjected to time-lapse microscopy to allow real-time investigation of processes such as vascular regression, sprouting and cellular plasticity. When combined with in vitro and in vivo models, as well as clinical data, this ex vivo PDR model will help in investigating patient responses based on particular sets of identifying markers, a step closer to the avenue for personalized medicine. Identifying patient-specific responses and/or response-specific markers is especially relevant in the case of PDR, which is a multifactorial disease with a complex interplay of microvascular, neurodegenerative, metabolic, genetic/epigenetic, immunological, and inflammation-related factors, thus requiring increasingly multidisciplinary efforts for development of improved therapeutic targeting and disease management. Besides whole-mount immunofluorescence, the ex vivo cultured FTs could also be retrieved from the fibrin by plasmin/nattokinase treatment and subjected to transcriptomic and proteomic analyses28. The suitability of the herein described model for ex vivo studies of fibrovascular tissues developed in other ocular conditions, such as in severe cases of sickle cell retinopathy, could also be explored in the future.
The authors have nothing to disclose.
The authors are most grateful to the medical and surgical retina colleagues, nurses and whole staff of the Diabetic Unit and Vitreoretinal Surgery Unit at the Department of Ophthalmology, Helsinki University Hospital for actively participating in the recruitment of patients. We thank Biomedicum Molecular Imaging Unit for imaging facilities. We thank Anastasiya Chernenko for excellent technical assistance. This study was supported by grants from the Academy of Finland (KL), University of Helsinki (KL), Sigrid Juselius Foundation (KL), K. Albin Johansson Foundation (KL), Finnish Cancer Institute (KL), Karolinska Institutet (KL), Finnish Eye Foundation (SL), Eye and Tissue Bank Foundation (SL), Mary and Georg C. Ehrnrooth Foundation (SL), and HUCH Clinical Research Grants (TYH2018127 after TYH2016230, SL), Diabetes Research Foundation (SL, KL, AK, EG) as well as the Doctoral Programme in Biomedicine (EG).
Material | |||
Microforceps | Medicon | 07.60.03 | Used for handling the FTs |
Disposable Scalpels – Sterile | Swann-Morton | 0513 | Used for FT dissection |
Culture dish, vented, 28 ml (60mm) | Greiner Bio-One | 391-3210 | Used for dissection and for testing fibrin gel formation |
Cell culture plates, 12-well | Greiner Bio-One | 392-0049 | Used for FT dissection and whole-mount immunofluorescence |
Reagent/centrifuge tube with screw cap, 15 mL | Greiner Bio-One | 391-3477 | |
Reagent/centrifuge tube with screw cap, 50 mL | Greiner Bio-One | 525-0384 | |
Millex-GV Syringe Filter Unit, 0.22 µm, PVDF | Millipore | SLGV033RS | Used to sterile-filter the fibrinogen solution |
Syringe, 10 mL | Braun | 4606108V | Used to sterile-filter the fibrinogen solution |
Polypropylene Microcentrifuge Tubes, 1.5 mL | Fisher | FB74031 | |
Cell-Culture Treated Multidishes, 24-well | Nunc | 142475 | Used for casting the FT/fibrin gels for native FT characterization and ex vivo culture |
Cell culture plates, 96-well, U-bottom | Greiner Bio-One | 392-0019 | Used for whole-mount immunofluorescence |
Round/Flat Spatulas, Stainless Steel | VWR | 82027-528 | Used for whole-mount immunofluorescence |
Coverslips 22x22mm #1 | Menzel/Fisher | 15727582 | Used for mounting |
Microscope slides | Fisher | Kindler K102 | Used for mounting |
Absorbent paper | VWR | 115-0202 | Used for mounting |
Name | Company | Catalog Number | Comments |
Reagents | |||
PBS tablets | Medicago | 09-9400-100 | Used for preparing 1x PBS |
Fibrinogen, Plasminogen-Depleted, Human Plasma | Calbiochem | 341578 | |
Hanks Balanced Salt Solution | Sigma-Aldrich | H9394-500ML | Used for preparing the fibrinogen and TA solution |
Fetal bovine serum | Gibco | 10270106 | Used for preparing the blocking solution |
Human Serum | Sigma-Aldrich | H4522 | Aliquoted in -20 °C, thaw before preparing the ex vivo culture media |
Gentamicin Sulfate 10mg/ml | Biowest | L0011-100 | |
Endothelial cell media MV Kit | Promocell | C-22120 | Contains 500 ml of Endothelial Cell Growth Medium MV, 25 mL of fetal calf serum, 2 mL of endothelial cell growth supplement, 500 μL of recombinant human epidermal growth factor (10 μg/ mL) and 500 μL of hydrocortisone (1 g/ mL) |
Sodium azide | Sigma-Aldrich | S2002 | Used for storage of the native and ex vivo cultured FTs. TOXIC: wear protective gloves and/or clothing, and eye and/or face protection. Use in fume hood. |
Acetone | Sigma-Aldrich | 32201-2.5L-M | Used to prepare the post-fixation solution. HARMFUL: wear protective gloves and/or clothing. Use in fume hood. |
Methanol | Sigma-Aldrich | 32213 | Used to prepare the post-fixation solution. TOXIC: wear protective gloves and/or clothing. Use in fume hood. |
Triton X-100 (octyl phenol ethoxylate) | Sigma-Aldrich | T9284 | Used for whole-mount immunofluorescence. HARMFUL: wear protective gloves and/or clothing. |
Hoechst 33342, 20mM | Life Technologies | 62249 | For nuclei counterstaining. HARMFUL: wear protective gloves and/or clothing, and eye and/or face protection. |
VECTASHIELD Antifade Mounting Medium | Vector Laboratories | H-1000 | Wear protective gloves and/or clothing, and eye protection. Use in fume hood. |
VECTASHIELD Antifade Mounting Medium with DAPI | Vector Laboratories | H-1200 | Mounting medium with nuclei counterstaining. Wear protective gloves and/or clothing, and eye protection. Use in fume hood. |
Eukitt Quick-hardening mounting medium | Sigma-Aldrich | 03989-100ml | TOXIC: Wear protective gloves and/or clothing, and eye protection. Use in fume hood. |
Thrombin from bovine plasma, lyophilized powder | Sigma-Aldrich | T9549-500UN | Dissolve at 100 units/ mL, aliquote and store at -20 °C, avoid repeated freeze/ thaw |
Aprotinin from bovine lung, lyophilized powder | Sigma | A3428 | Dissolve at 50 mg/ mL, aliquote and store at -20 °C, avoid repeated freeze/ thaw |
Name | Company | Catalog Number | Comments |
Growth factors | |||
Recombinant human VEGFA | R&D Systems | 293-VE-010 | 50 ng/ mL final concentration |
Recombinant human VEGFC | R&D Systems | 752-VC-025 | 200 ng/ mL final concentration |
Recombinant human TGFβ | Millipore | GF346 | 1 ng/ mL final concentration |
Recombinant human bFGF | Millipore | 01-106 | 50 ng/ mL final concentration |
Name | Company | Catalog Number | Comments |
Primary antibodies | |||
CD31 (JC70A) | Dako | M0823 | Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab |
CD34 (QBEND10) | Dako | M716501-2 | Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab |
CD45 (2B11+PD7/26) | Dako | M070129-2 | Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab |
CD68 | ImmunoWay | RLM3161 | Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab |
Cleaved caspase-3 (5A1E) | Cell Signalling | 9664 | Used at 1:200 dilution, Goat anti Rabbit Alexa 594 Secondary Ab |
ERG (EP111) | Dako | M731429-2 | Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab |
GFAP | Dako | Z0334 | Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab |
Ki67 | Leica Microsystems | NCL-Ki67p | Used at 1:1500 dilution, Goat anti Rabbit Alexa 594 Secondary Ab |
Lyve1 | R&D Systems | AF2089 | Used at 1:100 dilution, Donkey anti Goat Alexa 568 Secondary Ab |
NG2 | Millipore | AB5320 | Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab |
Prox1 | ReliaTech | 102-PA32 | Used at 1:200 dilution, Goat anti Rabbit Alexa 568 Secondary Ab |
Prox1 | R&D Systems | AF2727 | Used at 1:40 dilution, Chicken anti Goat Alexa 594 Secondary Ab |
VEGFR3 (9D9F9) | Millipore | MAB3757 | Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab |
α-SMA (1A4) | Sigma | C6198 | Used at 1:400 dilution, Cy3 conjugated |
Name | Company | Catalog Number | Comments |
Secondary antibodies | |||
Alexa Fluor488 Donkey Anti-Mouse IgG | Life Technologies | A-21202 | Used at 1:500 dilution |
Alexa Fluor594 Goat Anti-Rabbit IgG | Invitrogen | A-11012 | Used at 1:500 dilution |
Alexa Fluor568 Donkey anti-Goat IgG | Thermo Scientific | A-11057 | Used at 1:500 dilution |
Alexa Fluor568 Goat anti-Rabbit IgG | Thermo Scientific | A-11036 | Used at 1:500 dilution |
Alexa Fluor594 Chicken Anti-Goat IgG | Molecular Probes | A-21468 | Used at 1:500 dilution |
Name | Company | Catalog Number | Comments |
Microscopes | |||
Axiovert 200 inverted epifluorescence microscope | Zeiss | For imaging of the fresh and fibrin-embedded FT | |
SZX9 upright dissection stereomicroscope | Olympus | For FT dissection | |
LSM 780 confocal microscope | Zeiss | For imaging of whole-mount immunostained FT | |
AxioImager.Z1 upright epifluorescence microscope with Apotome | Zeiss | For imaging of whole-mount immunostained FT |