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30: Preparing for Printing
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The Tracing Image cannot be selected and moved the same way as other objects on your page. Instead, you must move the Tracing Image using menu commands. You have several options for adjusting the Tracing Image s position to better fit your design. First, you can align the Tracing Image with any object on your page by selecting the object and then choosing View Tracing Image Align with Selection. This lines up the upper-left corner of the Tracing Image with the upper-left corner of the bounding box of the object you ve selected. To precisely or visually move the Tracing Image to a specific location, choose View Tracing Image Adjust Position. Enter the x and y coordinates into their respective boxes in the Adjust Tracing Image Position dialog box, as shown in Figure 11-22. For more hands-on positioning, use the arrow keys to nudge the tracing AP element up, down, left, or right, one pixel at a time. Holding down the Shift key while pressing the arrow keys moves the Tracing Image in 5-pixel increments. Finally, you can return the Tracing Image to its default location of 9 pixels down from the top and 11 pixels in from the left by choosing View Tracing Image Reset Position. FIGURE 11-22
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8. In the Action Panel for the Submit button graphic, add an on (release) event and
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KeepDynamic.com/ANSI/AIM Code 39 Immunoblotting. Connexin-43 and keratins 3 and 12 are detected by immunoblotting of proteins from cell lysates with procedures described in Section 11.4. ABCG2 can be immune precipitated from cell lysates after cell surface biotinylation as described below. Antibodies used are the same as previously described. 11.3.3. Cryopreservation Brie y, for LSC cells, trypsinize, count, pellet by centrifugation, and resuspend the cells in freezing medium at 2 5 106 cells/mL with 1 mL per vial. Chill at a controlled rate of 1 C/h, using a commercial freezing box lled with isopropanol in a 80 C freezer overnight. On the next day, transfer the vials to liquid nitrogen. Freezing medium: 70% culture medium (DMEM/F-12), 20% FBS, 10% DMSO. Make fresh each time before freezing cells. 11.3.4. Variations and Applications of the Method LSC can be cultured as limbal explants or puri ed by FACS for side population cells. Explant Culture. After Dispase II digestion of human cornea (see Protocol 11.3), isolate the limbal epithelium and cut it into pieces about 1 mm in diameter. Seed these pieces on 3T3 feeder layers (see Protocol 11.2) or on amniotic membrane (see Protocol 11.1) with the epithelial side up. The cells will migrate out from the tissue over a period of 2 4 weeks.
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Operator = < > or @@ (Mac); < > (PC) > What It Means Means equal. The test is true if the items on both sides of the sign are exactly equal in value. For example, the statement 1 + 1 = 2 is true. Means not equal to. The test is true if the items on both sides of the sign are not equal in value. For example, Apple < > Banana is true. Means greater than. The test is true if the value of the item at the left of the sign exceeds the value of item to the right. For example, 3 > 2 is true. For text arguments, greater than means later in the standard collating sequence (for example, alphabetic order).
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Thus, to calculate the middle occurrence, you should divide by two and enclose the result in the Ceiling( ) function:
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12.6. CONCLUSIONS It is neither possible nor ethical to perform genetic engineering experiments in humans; thus development of human mammary cell culture techniques has been crucial and indeed necessary to locate, isolate, and characterize putative human mammary stem cells. Clearly, the models will need to be improved to even more accurately re ect the microenvironment so that existing and yet-to-be developed powerful culture techniques can be brought to bear on a number of new questions: How do stem cells integrate signals that control their functions and behavior What role does the microenvironment (other cells, hormones, growth factors, ECM molecules in short the niche) play in normal mammary gland morphogenesis and in the initiation and propagation of tumorigenesis How do stem cells relate to cancer What is the cellular etiology of different types of breast cancers It has been observed that histological subtypes in cancers emerge that appear lineage restricted, suggesting that stem and progenitor cells might lie at the cancer s origin. This scenario has been documented in the evolution of two distinct leukemias: Chronic myeloid leukemia is derived from hematopoietic stem cells, and acute myeloid leukemia is derived from committed granulocyte-macrophage progenitors [Daley, 2004; Jamieson et al., 2004]. Stemlike cells, isolated from a breast tumor, could be serially transplanted as a tumor in nude mice, but whether these were directly related to normal mammary stem or progenitor cells is not known [Al-Hajj et al., 2003]. It was additionally hypothesized that the stem cell microenvironment may play an important initiating or promoting role in tumorigenesis [reviewed in Bissell and Labarge, 2005]. Now that a hierarchy among mammary stem and progenitor cells has been identi ed for rodents and, more recently, for humans [Villadsen et al., 2007], as well as their respective locations within the mammary gland, a detailed analysis of their transformed phenotypes and what roles their microenvironments play in fostering speci c behaviors should take place. One particular challenge will be providing unequivocal proof that one normal stem cell does give rise to a mammary gland, and that the same cell, if transformed, gives rise to a tumor in vivo. Because 3D culture environments can model form and functions as complex as TDLU formation from single cells, they may also provide a malleable proving ground to demonstrate such a relationship. 12.7. SOURCES OF MATERIALS Item Anti-mouse IgG magnetic beads Ascorbic acid B27 supplement Bovine serum albumin (fraction V) Cholera toxin Collagenase Culture asks, 25 cm2 Dibutyryl cAMP Supplier Miltenyi Biotech Sigma Invitrogen (GIBCO) Sigma Sigma Sigma Nunc Sigma
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See 9 for implementation notes for a filter relationship using the essentials of this technique.
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