Visualization of actin in Xenopus explants
If colocalization with the actin cytoskeleton is required, we have determined that the following additional steps yield satisfactory results for simultaneous imaging of both ECM and integrins or cadherins Fig. 17.1 . 1. Following fixation as described above, explants are permeabilized with 0.5 saponin in TBS for 5 min. If both actin and ECM are to be visualized simultaneously, the permeabilization should be carried out before antibody incubation. 2. Wash three times at 5 min each with TBST as...
Isolation of mouse skin
To investigate the role of integrins for development and maintenance of the epidermis and the cycling and morphology of the HF, skin samples from various pre- and post-natal stages have to be collected and analyzed. Because skin morphology greatly differs by body region, skin samples must always be isolated from the same location e.g., back skin . The HF morphogenesis and the first postnatal catagen, telogen, and anagen development follow a rather precise time scale Table 12.4 , while later HF...
Imaging CellMatrix Interactions in 3D Collagen Gels
The use of MPLSM, which uses non-linear excitation to image a narrowly defined plane deep within tissue noninvasively, has the advantages of reduced phototoxicity Squirrell et al., 1999 and the ability to image deeper into turbid samples SHG Centonze and White, 1998 than other live cell imaging techniques. Moreover, its nonlinear excitation method is compatible with another nonlinear optical method second harmonic generation Campagnola et al., 1999 . Since collagen fibers are strong...
Culture in threedimensional matrigel
1. Detach the cells by trypsinization, and pipette gently to generate a single cell suspension. Wash the cells once with serum-free three-dimensional 3D culture medium DME HG F12 1 1 NEAA, 1 mg ml Neu YD 4-WT Neu YD 4-1355T W3
Cloning
Positive hybridoma culture wells are cloned via the following protocol. Two dilutions are made. The first is made so that the cells are diluted to 1 x 104 ml. Next, 100 ml of this cell suspension are further diluted to 10 ml in RPMI 1X AAT supplemented with 20 FBS and plated at 100 ml well in a 96-well plate at 1 cell well. Thymocyte feeder cells can be added at 1 x 105 per well or, as we have done on occasion, use Hybridoma Cloning Factor Bioveris Corporation 210001 at 10 . However, it is...
Preparation of reagents for the polyacrylamide gels
While the coverslips are being washed and siliconized, the reagents necessary for making the gels are prepared. These reagents include a saturated solution of acrylic acid N-hydroxy succinimide ester A-NHS in toluene, 10 ammonium persulfate APS , 40 acrylamide, and 1 bis-acrylamide. R- Si O -Si O -Si R I I I O O O Matri _NH2 GdV protein v 2 O N Figure 8.1 Reaction scheme forgenerating substrata of near physiological compliance. We generate substrata of near physiological compliance by...
Timelapse imaging of integrin trafficking in cells cultured on 2D surfaces
Considering recent interest and abilities in imaging cellular signaling events during cell adhesion and migration, the ability to also visualize integrin endocytosis and trafficking processes in live cells would prove to be highly informative. The use of commercially available kits that fluorescently label antibodies make this more readily possible. Direct labeling of integrin antibody sc-9970, mouse monoclonal IgG1 4B7R , Santa Cruz is carried out using a Zenon Alexa Fluor-555 mouse IgG1...
Selective expression of avb3 on angiogenic blood vessels
Endothelial cells grown in culture can bind vitronectin via av 3 or avb5, fibronectin via a5b1 or av 3, and collagen via al l or a2b1. However, avb3 is not detectable on endothelial cells lining normal blood vessels tn two. Instead, av 3 is highly expressed on bloodvessels near a tumor Brooks ei a ., 1994b , wound tissue Brooks ei a ., 1994a , rheumatoid arthritis Storgard ei a ., 1999 , or focal ischemia Okada ei a ., 1996 . Accordingly, anti-avb3 treatment blocks pathological angiogenesis but...
Evaluating angiogenesis in the zebrafish
Development of small molecule inhibitors requires innovative biological screens for selection of lead compounds. The zebrafish model is becoming widely used as a screening tool for drug discovery for several reasons. The statistical power is much greater due to the high numbers of embryos which can be generated with each mating. Assays are rapid finished within one week and toxicity data are accumulated quickly due to the sensitivity of the developing embryo. Importantly, screening compounds...
Purification and Analysis of Integrin Heterodimers
Integrins have a number of challenging biochemical properties that make their isolation in an active native state technically demanding. First, integrins are large proteins that form obligate noncovalent a b heterodimers, with both subunits being type I membrane proteins and thus are associated with membrane lipids Hynes, 1987 . Second, each subunit binds divalent cations, which is vital for ligand binding and for stability and association ofthe subunits Howard et al., 1982 . Third, each...
Preparation of washed platelets from mouse blood
Two methods are routinely being used for the isolation of blood from mice cardiac puncture and venepuncture. The main appeal to cardiac punctures is that they are not time consuming and do not require the use of anesthetics, because animals are euthanized through CO2 asphyxiation. On the other hand, because ofthe loss ofvasculature tonus in dead animals, blood has to be collected swiftly before platelets become activated. Cardiac punctures also present the disadvantage of yielding smaller blood...
CFSE staining of T cells
1. The sorted CD4 T cells are resuspended in PBS in a concentration of 1 X 107 ml. The CFSE stock solution is diluted 1 240 with PBS and added 1 50 to the cell suspension to reach a final concentration of0.5 mM. The mixture is briefly vortexed to ensure even distribution. The T-cell suspension is incubated for 10 min at RT in the dark. 2. The reaction is stopped by the addition of5 ml ofice-cold R10 medium. The cells are centrifuged at 300 Xg, and then taken up in R10 medium to obtain a final...
Preparation of gelfiltered platelets from human blood
An alternative to the washed platelet protocol is gel filtration of the PRP through a sepharose column. Gel-filtered platelets are typically more responsive to adenosine diphosphate stimulation than washed platelets, especially in the context of aggregation studies. PRP is prepared as described in paragraph 2.1.1. The sepharose 2B column is prepared as follows sepharose 2B GE Healthcare, Cat 17-0130-01 is poured into a 60-ml syringe containing a nylon mesh disc with a pore size less than 50 mm...
Preparation of the substrate 311 Treating glass
Much of the adhesion literature does not pay much attention to preparation of the substrate layer. Protein adsorption to glass or plastic substrates can vary in both efficiency and in the conformation of the adsorbed protein Garcia et al., 1999 Miller and Boettiger, 2003 . Glass coverslips are most convenient for the later counting of cells under a fluorescent microscope, the plastic coverslips are more uniform in performance. Our sources for both are given in Appendix 1. Glass coverslips have...
Spinning Disc Measurements
The spinning disc measures the mean shear stress required for cell detachment. For many cell types and conditions, we have found that the mean cell detachment force is proportional to the number of adhesive bonds Boettiger et al., 2001 Garcia and Boettiger, 1999 Garcia et al., 1998 Shi and Boettiger, 2003 . It has been argued that the cell would detach by a peeling mechanism. The adhesive bonds would detach sequentially, and hence there would not be a direct relationship between detachment...
Binding assays
1. Add 50 to 400 mg of cell lysate to 500 ml of buffer XT containing 3 mM of MgCl2. To this mixture, add 50 ml of the 10 v v suspension of integrin-coated beads described above , mix overnight at 4 . 2. Wash the integrin-coated resin twice with 1 ml ofbuffer XT to remove unbound proteins. 3. Elute bound proteins by resuspending the beads in SDS sample buffer and heating to 95 for 5 min. 4. Resolve samples on a 4 to 20 Tris-glycine SDS polyacrylamide gradient gel, and transfer to nitrocellulose...
Transfection adenoviral infection and siRNA
Spontaneously immortalized mouse embryo fibroblasts MEFs or NIH-3T3 cells are seeded the day before transfection at 3 x 105 cells 100-mm dish in DMEM containing 10 FBS without antibiotics. The morning of transfection, 5 mg of DNA are added to 750 ml DMEM for each transfection. Twenty microliters of Lipofectamine Plus reagent Invitrogen is added to the DNA-DMEM solution and allowed to form a complex at room temperature for 15 min. Thirty microliters of Lipofectamine reagent Invitrogen is diluted...
EnDOtheLiAL CeLLSpeciFic InteGRin a4 DeLetion MutAnt
To evaluate the role of integrin a4 1 in lymphangiogenesis, C57BL 6 x 129 mice homozygous for the floxed a4 allele Scott et al., 2003 were crossed to C57BL 6 mice expressing Cre under the control of the Tie2 promoter B6. Cg-Tg Tek-cre 12Flv J from Jackson Labs mice to generate Tie2Cre a 4 flox mice. Alternatively, C57BL 6 x 129 mice homozygous for the floxed a 4 allele were crossed to C57BL 6 mice expressing Cre under the control of the VE-Cadherin promoter Alva et al., 2006 to generate Tie2Cre...
Yuliya Pylayeva Wenjun Guot and Filippo G Giancotti
2. Experimental Approach 442 3. Measurement of Primary Tumor Growth 443 4. Spontaneous and Allograft Lung Metastasis Assays 444 5. Analysis of Tumor Sections 448 5.1. Methods 450 6. Detergent Extraction of Tumor Samples 452 7. Ex Vivo Culture of Mammary Tumor Cells 452 7.1. Method 453 8. Genetic Modification of Primary Mammary Tumor Cells 453 8.1. Production of retroviral lentiviral stocks and cell infection 9. Disruption of Epithelial Adhesion and Growth Control 456 9.1. Cell proliferation...
Imaging of adhesion dynamics
Advances in live fluorescence cell imaging have continued to improve our knowledge ofthe molecular architecture and dynamics ofadhesive complexes Ezratty et al., 2005 Webb et al., 2004 Zaidel-Bar et al., 2004 . Focal complexes are early adhesions that form at the cell periphery, and are more highly dynamic than focal adhesions. Focal adhesions, in contrast, are localized at the end of actin stress fibers at both central and peripheral regions of the cell, and contain some of the same components...
Basic configuration and fabrication of the window chamber
The window chamber apparatus, shown in Fig. 16.2, consists of two complementary titanium alloy frames, each with a 1.2-cm diameter opening, and on one ofthe frames the opening is fitted with a titanium ring for attachment of the glass coverslip. The ring extends 0.025 in 0.6 mm to the tissue to Figure 16.2 The mouse dorsal skinfold window chamber apparatus. The schematic drawing depicts two complementary titanium plates. The plates are held together with screws, nuts, and sutures. Figure 16.2...
Generation of EB precursors Procedure
1. The ESC medium is removed and the culture dish with ESCs is rinsed twice with 10 ml of prewarmed 37 PBS. 2. The ESC colonies are detached from feeders by adding 3 ml of 0.25 Trypsin 0.53 mMEDTA for 2 to 3 min at room temperature RT . Cell detachment should be monitored under the microscope and can be facilitated by tapping the tissue culture dish. 3. When ESC colonies start to separate from feeder cells, the trypsin is immediately inactivated by adding 6 ml of feeder cell medium. 4. After...
References Vvd
Alonso, L., and Fuchs, E. 2006 . The hair cycle. J. Cell Sci. 119, 391-393. Aumailley, M., Pesch, M., Tunggal, L., Gaill, F., and Fassler, R. 2000 . Altered synthesis of laminin 1 and absence of basement membrane component deposition in b1 integrin-deficient embryoid bodies. J. Cell Sci. 113, 259-268. Barnden, M. J., Allison, J., Heath, W. R., and Carbone, F. R. 1998 . Defective TCR expression in transgenic mice constructed using cDNA-based a- and b-chain genes under the control of heterologous...
Procedure Preparation oftwodimensional collagen gels
1. Two-dimensional collagen gels are prepared by mixing 800 ml ofchilled, acid-soluble type I collagen 4 mg ml with 100 ml of 10x M199 pH 7.4 media, and 30 ml of bicarbonate and 70 ml of ddH2O. As with preparing a 3D gel culture, the ingredients are mixed thoroughly on ice, with care taken to avoid introducing bubbles into the gel. 2. The collagen can then be transferred to a 35-mm dish modified with a coverslip bottom Electron Microscopy Sciences 7670-02 . While regular tissue culture dishes...
Spontaneous and Anograrr Lung Metastasis Assays
The mouse models we utilize in the laboratory MMTV-Neu YD and MMTV-PyMT develop mammary carcinomas that frequently metas-tasize to the lung, allowing an examination of the effect of integrin signaling on the multiple steps of the metastatic cascade over the natural time course of disease progression in vivo. We have compared the extent of spontaneous pulmonary metastases in Neu YD b4-WT and Neu YD b4-1355T mice carrying primary tumors of comparable size Fig. 19.1 . Analysis of hematoxylin and...
IntravitaL Microscopy of Lymph Nodes
To image lymphatic vasculature, lymphangiogenesis, and metastasis within lymph nodes, dorsal skinfold window chambers can be implanted into mice of varying backgrounds over lymph nodes. Mice are anesthetized with ketamine 100mg kg and medetomidine 250 mcg kg injected intraperitoneally. The entire dorsum is swabbed with Betadine, and then 70 ethanol is used to swab the dorsum clean. All surgical instruments and gauze are sterilized prior to use, and a bead sterilizer used to re-sterilize...
Cell lines
PAC-1 POW-2 binding Each reaction is performed on 0.5 to 7 x 106 cells in Walsh's buffer. For each sample, 50 ml of cell suspension are incubated with or without 5 ml of an agonist and or inhibitor for 10 min at room temperature. Control samples are treated with 5 ml of 100 mM EDTA, pH 7.35, or a specific aIIbb3 antagonist, such as 10 of mM integrilin or 2 mM of RGDS to estimate nonspecific binding , or with 5 ml of 10 mM MnCl2 to stimulate aIIbb3 extrinsically. PAC-1 or POW-2 is added to each...
In vitro models to assess vascular permeability
The process of vascular permeability has extreme consequences in vivo, since leak of blood and plasma proteins can quickly damage tissues at sites of leak. In vitro, permeability can be assessed by examining the integrity of an endothelial cell monolayer in the presence or absence ofpermeability factors and or test agents. Commonly used in vitro models include the ''transwell assay,'' in which an endothelial cell monolayer is grown to confluency on a Boyden chamber or transwell . Tracers of...
Mouse strain and myeloma
There are many protocols for the production of monoclonal antibodies Harlow and Lane, 1988 Howard and Bethell, 2001 Kohler and Milstein, 1975 Oi and Herzenberg, 1980 Peters and Baumgarten, 1992 . The method that we have used for the past 15 years is based on the protocol described by Oi and Herzenberg 1980 , and makes use of the Robertsonian Rb 8.12 5 Bnr mouse strain RBF DnJ , and the FOX-NY myeloma cell line described by Taggart and Samloff 1983 . FOX-NY was isolated as a mutant of the NS-1...
LacZ staining on cryosections
As already introduced in Section 3.3, the bacterial lacZ gene can be engineered into the targeting construct so that it is activated upon Cre-mediated gene disruption. Consequently, it can then be used to examine the expression of the disrupted gene. This approach was used in our laboratory to monitor the K5-Cre-driven excision of the 1 integrin gene Brakebusch et al., 2000 . The protocol described below uses the -gal substrate X-gal that produces blue precipitates that are insoluble in fixed...
Chick chorioallantoic membrane angiogenesis assay
The chorioallantoic membrane CAM in the chicken embryo has been used since the early 1900s to study angiogenesis in vivo see the short commentary by Ribatti, 2004 . Many years later, Cheresh and colleagues employed this model to demonstrate the requirement of av 3 for angiogenesis induced by VEGF, while that induced by bFGF requires av 5 Brooks et al., 1994a Friedlander et al., 1995 . This model is still being used routinely as a screen for integrin antagonists and antiangiogenic agents....
Genetic ModiFiCATioN of Primary Mammary Tumor Cells
The derivation of primary cell cultures from tumors provides invaluable opportunities to study oncogenic signaling pathways. Furthermore, the cells can be genetically modified by infection with viral vectors encoding interfering RNA sequences or various cDNAs. However, since the MMTV promoter is regulated by hormones Ucker et al., 1981 , its activity tends to decline once primary cells have been explanted in culture even if the cultures are supplemented with hydrocortisone . It is therefore...
The Miles assay to assess leak in the skin
Vascular leak in the skin can be evaluated using the Miles assay, developed in 1952 Miles and Miles, 1952 . The vascular permeability properties of permeability agents such as VEGF can be determined by leakage of Evan's blue dye a common marker for vascular leakage after local intradermal injection. This response can be assessed in normal mice, mice with gene-targeted deletions, or mice treated with potential inhibitors of the vascular leak response Eliceiri et al., 1999 . Importantly, this...
Immunostaining
Coverslips are blocked in 10 serum PBS for 30 min at room temperature. The serum is preferentially from the same animal species as the one that the primary detecting antibody was raised in. Coverslips are then incubated with the primary antibody at 1 100 to 1 1000 in 10 serum-PBS for 45 min at 37 , washed three times with PBS, and incubated for 45 min at 37 with fluorophore-labeled secondary antibody Invitrogen and Jackson Immuno-chemicals at 1 500 to 1 1000 in 10 serum-PBS. For staining of...
The activation and functions of Src kinase
Src kinase belongs to the non receptor tyrosine kinases family that consists of, sequentially, an N-terminal SH4 domain, a unique region, a SH3 domain, an SH2 domain, a catalytic domain, and a C-terminal regulatory sequence Thomas and Brugge, 1997 . In the inactive state, the SH3 and SH2 domains of Src kinase are coupled together by intramolecular interactions, and the catalytic kinase domain of Src is masked by its interaction with the C-terminal tail, thus preventing its action on substrate...
Identification of Integrin Knockout Phenotypes Based on Educated Guesses from
Many of the roles that have been suggested for integrins based on studies with primary cell lines or cultured cells have not been validated in integrin knockout mice. One important use ofthese mice can be identifying which of the many suggestions made from in vitro studies actually have in vivo relevance. In a few notable cases, clues from in vitro studies have been critical in identifying knockout phenotypes that would not otherwise have been apparent. Two examples from b5 subunit knockout...
Preparation of Specimens for NegativeStain EM
Our laboratory uses two types of transmission EM negative-stain EM Hayat and Miller, 1990 , in which macromolecular complexes are surrounded by a heavy metal stain Fig. 15.2 , and cryo-EM Fig. 15.3 , in which protein complexes are suspended in a vitrified buffer Chiu, 1993 Figure 15-2 Negative-stain electron micrograph of the soluble ectodomain of avb gt The molecules are randomly distributed making recognition of views difficult. Two putative side views are circled, showing the characteristic...
Distinct functions for avb3 and avb5 during angiogenesis and vascular leak
While avb3 recognizes several different ligands, avb5 primarily binds to vitronectin. In carcinoma cells, av 3 is detected in focal contacts colocaliz-ing with vinculin, talin, and the ends of actin filaments, while avb5 shows a distinct, nonfocal contact distribution on the cell surface Wayner et al., 1991 . Thus, two homologous vitronectin-binding integrins can be distributed differentially on the cell surface and likely direct distinct cellular processes. In fact, studies using...
References Xru
Abrams, M. J., Juweid, M., Tenkate, C. I., Schwartz, D. A., Hauser, M. M., Gaul, F. E., Fuccello, A. J., Rubin, R. H., Strauss, H. W., and Fischman, A.J. 1990 . Technetium-99m-human polyclonal IgG radiolabeled via the hydrazino nicotinamide derivative for imaging focal sites of infection in rats. J. Nucl. Med. 31, 2022 2028. Agrez, M., Chen, A., Cone, R. I., Pytela, R., and Sheppard, D. 1994 . The alpha v beta 6 integrin promotes proliferation of colon carcinoma cells through a unique region of...
Eric A Klein Yuval Yung Paola Castagnino Devashish Kothapalli and Richard K
2. Preparative Methods 156 2.2. Transfection, adenoviral infection, and siRNA 158 2.3. Preparing substrata of near physiological compliance using matrix proteins linked to polyacrylamide gels 162 3.1. Cell cycle analysis by propidium iodide staining and 3.2. Crystal violet staining to monitor cell proliferation 165 3.4. Rho family GTPase pulldown assays 168 3.5. Assays for transcriptional regulation 169 3.6. Special analytical procedures needed for experimentation on acrylamide-extracellular...
Production of monoclonal antibodies to integrin receptors using hybridoma
Our primary protocol for the production of an antibody to an integrin receptor is summarized in the flow chart in Fig. 7.1. However, first the antigen, the test system, and the immunization and fusion strategy must be chosen. We decided for the purposes of this paper to make a function-blocking mouse monoclonal antibody to the human a6 integrin receptor. The decisions we made in order to accomplish this and the reasons for each decision will be described in detail. Data have been recorded over...
MiGration Assays
To determine whether specific integrins regulate LEC migration, cell migration assays were performed using Costar Transwells. The undersides of 8-mm transwell inserts were coated with fibronectin, CS-1 fibronectin, or vitronectin 5 mg ml for 12 h at 4 . Nonspecific binding sites were blocked by incubation with 3 BSA in PBS for 1 h at 37 . Cells were resuspended in migration buffer Hepes-buffered M199 medium containing 1 BSA, 1.8 mM CaCl2, 1.8 mM MgCl2, and 0.2 mM MnCl2, pH 7.4 , and 50,000...
Methods in Enzymology
Volume I. Preparation and Assay of Enzymes Edited by Sidney P. Colowick and Nathan O. Kaplan Volume II. Preparation and Assay of Enzymes Edited by Sidney P. Colowick and Nathan O. Kaplan Volume III. Preparation and Assay of Substrates Edited by Sidney P. Colowick and Nathan O. Kaplan Volume IV. Special Techniques for the Enzymologist Edited by Sidney P. Colowick and Nathan O. Kaplan Volume V. Preparation and Assay of Enzymes Edited by Sidney P. Colowick and Nathan O. Kaplan Volume VI....
Transient transfection
Since it is not always necessary or unproblematic to create a stably expressing cell line, transient transfection of the desired construct may be utilized. However, due to the low transfection efficiency for breast epithelial cells typically 20 to 30 a fluorescent marker, such as GFP, is necessary for cell biology experiments. For transient transfection, seed x 105 MEFs or MDA-MB-231 cells into 12-well culture plates 2 days prior to imaging. After 24 h, or after the cells are more than 80...
Live imaging of FN fibrils
For live imaging of FN fibrils, the mAb 4H2 a non-function-blocking antibody raised against the central cell binding domain of frog fibronectin Ramos and DeSimone, 1996 was conjugated to the Cy3 fluorophore using an NHS-ester form of Cy3 FluoroLink, Amersham Pharmacia Biotech . Cy3-4H2 was dialyzed against DFA without BSA and concentrated with cutoff filters Centricom YM-30 centrifuge filters, Amicon . Confocal time-lapse sequences were collected using a confocal laser scan head PCM2000 Nikon,...
Live Imaging of Fibronectin Fibrils
We have developed a novel preparation that enables the dynamic assembly and remodeling of FN fibrils by ectodermal and mesodermal tissues to be visualized Fig. 17.3 . One particular advantage of this Figure 17.3 A Explants with epithelial cell layers were prepared from late blastula or early gastrula stage embryos. B Explants were sandwiched between a thin lower and thick upper sheet of agarose, and a glass coverslip bridge held in place by silicone grease. The thin sheet of agarose typically...
Preparation of thin agarose sheets
A heat block is preheated to 60 and filled with water. One-percent agarose in double-distilled water is prepared and kept molten in the heat block. A single large coverslip is then placed on the heat block and a single drop of agarose pipetted onto the warmed coverslip, followed immediately by a smaller coverslip fragment. The thickness ofthe resulting agarose gel can be controlled by the amount of molten agarose sandwiched between the two glass coverslips. The coverslip with the agarose is...
Rho Activity Assay from Cells Cultured in 3D Collagen Gels
Recently we have been able to optimize the G-LISA RhoA activation assay luminescence based from Cytoskeleton, Inc. Denver, CO to analyze RhoA activity using breast epithelial cells cultured within 3D collagen gels. In the past the only way to analyze the activity of RhoGTPase was to perform a very labor-intensive Rho pull-down assay. While a very reproducible Rho pull-down protocol was developed in our lab to assay breast epithelial cells in 3D collagen gels Wozniak and Keely, 2005 , the assay...
Rho activity assay
Although Rho pull-downs have traditionally been performed in solution using GST-Rhotekin, we currently use an ELISA-based kit G-LISA Cytoskeleton to measure Rho activation. Cells are seeded in six-well dishes one well sample so that after starvation they are 50 confluent 10 to 20 x 104 cells . Measurement of Rho activity is done according to the G-LISA protocol with the supplied reagents. Briefly, cells are collected and lysed. A portion 10 of the lysate is removed to measure protein...
Murine Lymphangioma Model
Lymphangiogenesis in the context of inflammation was modeled in mice by injecting C57B1 6 mice, 6 weeks of age, twice at 15-day intervals in the intraperitoneal cavity with 200 ml of incomplete Freund's adjuvant incomplete incomplete Freund's Freund's adjuvant adjuvant incomplete incomplete Freund's Freund's adjuvant adjuvant Figure 18.7 Lymphangioma mouse model. A Murine lymphangioma was induced by injection of incomplete Freund's adjuvant in the peritoneal cavity at 2-week intervals. B Mice...