This work was supported by US Department of Defense (BC075554 to B.G. and BC061403 to D.K.), US National Institutes of Health (U54GM064346 to J.v.R.; CA100324 to J.C., J.E.S. and J.W.; CA77522 to J.E.S., U54CA126511 to J.C. and B.G.). We thank D. Entenberg for help with microscopy, M. Rottenkolber for help in fabrication of the imaging box and J. Pollard (Albert Einstein College of Medicine) for the F4/80 antibody.

1. Generation of fluorescent tumors using injection of tumor cells into the mammary fat pad:
<ol>
<li>Grow a cell line expressing Dendra2 protein (as a cytoplasmic marker) to 40 - 80% confluency.</li>
<li>Rinse dishes at least 3 times with PBS w/o Ca2+ or Mg2+.</li>
<li>Add 3 ml of trypsin per 10 cm dish and incubate at 37 &deg;C until most of the cells detach and then hit the dish against a flat surface to shake it.</li>
<li>Rinse all the cells off the dish, and use a scraper (rubber policeman) to collect matrix as well.&nbsp; Add the 5 ml of PBS.&nbsp;Take an aliquot to count during centrifugation.</li>
<li>Centrifuge at 800 g for 5 min.</li>
<li>Aspirate and resuspend in PBS to a concentration of 5- 10x 106/ml.&nbsp; Store on ice until injected (inject within 30 min).</li>
<li>Place a cage containing 4-5 week old female immune deficient mice (e.g. SCID) inside a sterile hood.</li>
<li>Spray the area around the 4th (abdominal) nipple with 70% ethanol.</li>
<li>Inject 0.1 ml into mammary fat pad. If an assistant can be found, one person can hold the mouse in place while the other inserts the needle inside the mammary fat pad. If you are injecting by yourself, put the mouse under light anesthesia using isoflurane.</li>
<li>Once the tumor has grown to 5-7 mm, the Mammary Imaging Window (MIW) should be inserted. Note: Alternatively, for some applications, the MIW can be inserted on top of the healthy mammary fat pad and the cells can be injected afterwards. That approach allows imaging of transiently transfected cells in a physiological environment.</li>
</ol>
2. Manual fabrication of the Mammary Imaging Window (MIW):
<ol>
<li>MIWs (Figure 1A) are made of tissue grade plastic to guarantee biocompatibility. For manual construction, we use 5 or 10 cm dishes.</li>
<li>Put latex gloves on.</li>
<li>Heat up a tissue-culture dish and creat a curved surface by pushing a rounded, hard object against the heated dish (we use a 1-inch dremmel bit for this).</li>
<li>Cut out the center of the dish using a heated razor blade. </li>
<li>Use a small, cone-shaped dremmel bit to make a hole in the center of the dome-shaped plastic base (6-7mm in diameter). </li>
<li>Make the edges of the plastic base completely smooth by sanding it with the dremmel and further filing it. </li>
<li>File the top of the plastic base making a flat surface for the glass coverslip. The diameter of the filed, flat surface should be 9-10mm.</li>
<li>Glue the 8mm circular glass coverslip (number 1) on the flattened surface using the superglue (cyanoacrylate adhesive).</li>
<li>Wait for the glue to dry (15 minutes).</li>
<li>Use a heated 26G needle to make eight suturing holes puncturing from the outside (where the glass is) to the inside of the base. Holes should be evenly distributed around the coverslip, .5-1mm away from the edge of the coverslip. </li>
<li>Widen the holes using a 5-0 suturing needle.</li>
<li>Puncturing holes in the plastic base will make the inner surface uneven. Using sand paper, make this surface completely smooth.</li>
<li>Change the gloves and brush of small plastic particles from the MIW using a small brush.</li>
<li>Wash the MIW with deionized water.</li>
<li>Wash the MIW using 70% ethanol. Use a Q-tip to clean the glass so it is completely transparent. If there are foggy spots present from the glue vapor, carefully use acetone with a Q-tip.</li>
<li>Sterilize the MIW by UV exposure for 3 h at each side.</li>
</ol>
Semi-Manual fabrication of the Mammary Imaging Window (MIW):
In order to fabricate the plastic base, we are currently using silicone rubber casting molds created using hand-made MIWs. The mold is comprised of two parts of silicone rubber that make an exact replica of both the front and back of the original when filled with polyester resin and then immediately joined together.&nbsp;The liquid polyester resin is mixed together 9:10 and results in a hard structure when fully cured in 48h. The plastic is UV protected and archival without breaking down or becoming yellow over time. After the base is cured, steps 2.8-2.16 are done the same way as for manually-made MIWs.
3. Insertion of the MIW:
<ol>
<li>The area around the 4th nipple should have a small (5-7 mm diameter) tumor several days/weeks after injection of tumor cells depending on the cell type (Figure 1B). How much time has passed after injection of cells expressing Dendra2 photoswitchable protein or other fluorescent proteins depends on the cell line used. The tumor should not be visibly necrotic, and should have an intact skin with hair on top of the tumor. Mice with necrotic tumors should be euthanized.</li>
<li>Prepare the sterile space for the surgery- lay down a piece of sterile cloth inside a sterile hood. Put a piece of sterile gauze in the middle, and lay a few MIWs and sterile instruments around it: we use small standard scissors, spring scissors, microdissecting tweezers, microdissecting forceps, needle holder. Keep sterile Q-tips, a bottle of 70% ethanol and sterile gloves in the corner of the hood.</li>
<li>The mouse is anesthetized in the sterile hood using IP injection of 2.5% avertin (20 &mu;l/g) in HBSS or, alternatively 10 &mu;g/kg ketamine + 10 &mu;g/g xylazine (contact your Animal Care Institute for approvals and ordering). Note: Avertin solution should be prepared bi-weekly, filter sterilized using a 0.22 &mu;m filter and stored as 500 &mu;l aliquots at 4 &deg;C (in the dark).</li>
<li>Remove hair by shaving the area above the tumor using a small animal shaver.</li>
<li>Remove the rest of the hair using Nair hair removal cream (available in any drugstore). Clean the skin using ethanol-dipped Q-tips.</li>
<li>Transfer the animal onto the sterile gauze and apply ophthalmic ointment to the eyes to keep them from drying and infection.</li>
<li>Sterilize the skin with betadine and clean with 70% ethanol.</li>
<li>Transfer the animal onto a sterile surgical cloth inside the sterile hood. </li>
<li>Pull the skin immediately medial to the nipple using forceps and cut ~2mm incision in the skin.</li>
<li>Separate the underlying mammary fat pad from the skin using dissection scissors and forceps. The skin and hole stretch during this separation step to a size that will accommodate insertion of the MIW.</li>
<li>If accidently a vessel is hit during sugery, use a sterile Q-tips to remove any blood that is produced/to stop the bleeding from becoming excessive. </li>
<li>Insert the MIW such that there is skin on top of the MIW base and suture in place using non-absorbable thread and reverse cutting needle.</li>
<li>Use tissue adhesive or cyanoacrylate to fill in the suturing holes and secure the MIW to the skin.</li>
<li>Add TMP-SMX antibiotic mix: (Sulfamethoxazole 0.6 mg/ml, Trimethoprim 0.12 mg/ml) into the cage water bottle for 3 days before and after the surgery. </li>
<li>The animal stays under anesthesia for 1-4h after avertin IP injection. Since the surgery commonly takes ~45min, it is important to help the animal recover afterwards:</li>
<li>Place a hand heating pad (37 &deg;C) (available in drugstores) on the bottom of the cage and cover it with gauze.</li>
<li>Keep the animal on top of the pad until it turns on its stomach and starts walking.</li>
<li>If the animal is unconscious for more than 3h, inject 0.3 ml of HBSS intraperitoneally for rehydration.</li>
<li>The animal is allowed to recover over the next 3-4 days before the first imaging session takes place.</li>
</ol>
4. Imaging Box Construction
The imaging box assures that the MIW is sitting flat, right above the microscope objective. It also facilitates temperature and anesthesia control through constant airflow of isoflurane. The box is made of plexiglass and glued together using plastic weld. It is fitted for the specific microscope stage and therefore the shape of its bottom varies depending on the microscope used (Figure 2 shows the scheme of the box fitted to Leica SP5 microscope stage). Dimensions of the box in centimeters are l=11.4 cm, w=7.6 cm, h=4.4 cm (Figure 2A). The bottom of the box consists of a hollow plate, 12.7x8.4 cm in size, which fits inside the Leica SP5 microscope stage, and two sliding &ldquo;doors&rdquo;, 5.7 X 5.7 cm_each, which form a circular opening (d=2.22 cm) when closed. The MIW fits into this opening. The front piece contains the inlet hole for the anesthesia delivery while one of the side pieces contains an outlet hole which leads to the vacuum. Note that the condenser and the slide holder need to be removed prior to the imaging box placement.
5. Imaging Box Use
<ol>
<li>Place the animal under anesthesia with isoflurane and apply ophthalmic ointment to the eyes.</li>
<li>Attach the isoflurane exhaust from the anesthesia machine to the imaging box front, on the outside of the inlet hole (Figure 2B).</li>
<li>Attach a second tube to the outlet hole of the imaging box. This tube should be attached to the gas filter and then to the vacuum. </li>
<li>Open the box lid and open the box doors and place the animal, MIW side facing the bottom, and sliding the doors into the box.</li>
<li>Hold the box and adjust the bottom doors so that the MIW base is held and immobilized between them. The MIW base should be at the same level as the imaging box doors, while the MIW coverslip should be just a few millimeters below this level. Make sure that the positioning of the MIW between the bottom sliding doors ensures that the coverslip (glass portion of the MIW) lies flat, parallel to the bottom doors the box. This will ensure proper focusing. </li>
<li>Lower the microscope objective, to allow space for placement of the box on top of the microscope stage.</li>
<li>Place the box on the microscope stage, and adjust the stage so that the MIW coverslip is above the objective. </li>
<li>Focus the objective and start imaging.</li>
<li>While using isoflurane, the animal should be monitored by visual inspection of the breathing frequency. This can be done visually, or by monitoring the frequency of breathing artifacts appearing during imaging collection (these can interfere with data acquisition). The MouseOx pulse oximetry system (Starr life Sciences Corp) has been used succesfully. That way the data can be gathered continuously without turning the lights on in the room to check on the animal. Note: After each imaging session, animal recovery should be facilitated by wrapping a small hand heating pad in gauze or kimwipes and putting it under the animal in the cage. Note: If an immersion objective is used to image through the MIW, the immersion medium (glycerol, water, oil) should be cleaned off the imaging window. The MIW should be inspected for any cracks or other damage that might have occurred during imaging. This is critical for collecting data during multiple imaging sessions. </li>
</ol>
6. Photoswitching and imaging of Dendra2-labeled cells
The procedure is described for the Leica SP5 confocal microscope; the laser power at the focal plane, available objectives and software options vary when using other confocal microscopes or multiphoton imaging set up, and therefore the protocol described below should be used as a guide to optimize the experiment on your microscope.
<ol>
<li>By observing the tumor through the eyepiece (10x) with the green fluorescence filter, locate major blood vessels which are visibly flowing and laying flat in the same focal plane.</li>
<li>Position one of the vessels in the center of the field and switch to PMT detection.</li>
<li>Set up sequential imaging collection for two channels. One of the channels uses 488nm laser line (10% power), and collects scattering from extracellular matrix (480-495nm) and emission from the green form of Dendra2 (505-540nm). The second channel uses the 543nm laser line (90% power) and collects emission from the red form of Dendra2 (555-600nm). Collect 3D images pre-photoswitching.</li>
<li>Use the ROI scan option to photoswitch a chosen population of cells using the 405nm laser line (30% power, 20-40 scans). Collect emission of the red form of the protein to monitor the switching.</li>
<li>Using the same sequential routine as in 6.3, collect post-switching 3D images.</li>
<li>If photoswitching more than one region in the tumor, it is advised to take pictures through the oculars using a digital camera, in both green and red channels (Figure 3A, B). This will help in the orientation during the subsequent imaging sessions. Note: Additionally, blood vessels can be temporarily labeled using tail-vein injection of fluorescent dextran (Cascade Blue or Alexa Fluor 647, both 10kDa). A few hours after the injection, dextrans will leave the blood vessels and permanently label the macrophages.</li>
</ol>
Representative Results:
Figure 3C shows a rectangular photoswitched region (red) oriented orthogonally relative to the blood vessel (no fluorescence). Non-photoswitched cells are green, while the scattering from the extracellular matrix is purple. Image is a maximum intensity projection of four images along the Z-axis (20-50 &mu;m depth).

<ol>
	<li>Farina, K. L., Wyckoff, J. B., Rivera, J., Lee, H., Segall, J. E., Condeelis, J. S., Jones, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+motility+of+tumor+cells+visualized+in+living+intact+primary+tumors+using+green+fluorescent+protein.">Cell motility of tumor cells visualized in living intact primary tumors using green fluorescent protein.</a> Cancer Res. 58, 2528-2532 (1998).</li><li>Naumov, G. N., Wilson, S. M., MacDonald, I., Schmidt, E. E., Morris, V. L., Groom, A. C., Hoffman, R. M., Chambers, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+expression+of+green+fluorescent+protein,+coupled+with+high-resolution+in+vivo+videomicroscopy,+to+monitor+steps+in+tumor+metastasis.">Cellular expression of green fluorescent protein, coupled with high-resolution in vivo videomicroscopy, to monitor steps in tumor metastasis.</a> J Cell Sci. 112, 1835-1842 (1999).</li><li>Brown, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+gene+expression,+angiogenesis+and+physiological+function+in+tumors+using+multiphoton+laser+scanning+microscopy.">In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy.</a> Nature Med. 7, 864-868 (2001).</li><li>Wang, W., Wyckoff, J. B., Frohlich, V. C., Oleynikov, Y., Huttelmaier, S., Zavadil, J., Cermak, L., Bottinger, E. P., Singer, R. H., White, J. G., Segall, J. E., Condeelis, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+behavior+in+metastatic+primary+mammary+tumors+correlated+with+gene+expression+patterns+revealed+by+molecular+profiling.">Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling.</a> Cancer Res. 62, 6278-6288 (2002).</li><li>Sidani, M., Wyckoff, J., Xue, C., Segall, J. E., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+microenvironment+of+mammary+tumors+using+multiphoton+microscopy.">Probing the microenvironment of mammary tumors using multiphoton microscopy.</a> J Mammary Gland Biol Neoplasia. 11, 151-163 (2006).</li><li>Wyckoff, J. B., Wang, Y., Lin, E. Y., Li, J. F., Goswami, S., Stanley, E. R., Segall, J. E., Pollard, J. W., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+macrophage-assisted+tumor+cell+intravasation+in+mammary+tumors.">Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors.</a> Cancer Res. 67, 2649-2656 (2007).</li><li>Egeblad, M., Ewald, A. J., Askautrud, H. A., Truitt, M. L., Welm, B. E., Bainbridge, E., Peeters, G., Krummel, M. 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<td>PBS w/o Ca, Mg</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14190-144</td>
<td>&nbsp;</td>
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<td>Trypsin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25300</td>
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<td>HBSS</td>
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<td>Gibco</td>
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<td>DMEM media (MDA-MB-231 cells)</td>
<td>&nbsp;</td>
<td>Gibco</td>
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<td>Isoflurane(Aerrane)</td>
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dendra2 photoswitching through the mammary imaging window

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells5, 6, angiogenesis3 and immune cells response7-9. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice10. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra211 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.

Watch this Scientific Journal Video about Dendra2 Photoswitching through the Mammary Imaging Window at JoVE.com

Dendra2 Photoswitching through the Mammary Imaging Window

Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4  has resulted in important insights into ...

dendra2-photoswitching-through-the-mammary-imaging-window

Research

JoVE Journal

Biology

11.6K Views.  Albert Einstein College of Medicine - Yeshiva University. Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.

Video: Dendra2 Photoswitching through the Mammary Imaging Window

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.