Name: longitudinal intravital microscopy using a mammary imaging window with replaceable lid
Uploaded: 2022-01-20T13:00:00
Duration: 10 min 53 s
Description: Watch this Scientific Journal Video about Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid at JoVE.com

This work was supported by the Research Foundation Flanders (PhD grant fundamental research 11L7222N to M.C.), the Boehringer Ingelheim Foundation (PhD Fellowship to C.L.G.J.S), an EMBO postdoctoral fellowship (grant ALTF 1035-2020 to C.L.G.J.S.), and the Doctor Josef Steiner Award (to J.v.R).

<ol>
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The authors have nothing to disclose

The R.MIW allows longitudinal imaging of the healthy and diseased mammary gland in its native&#160;and minimally disrupted environment and permits repeated visualization of the mammary gland at diverse developmental stages. The R.MIW design allows the window to be opened at any point during the experiment. Long-term visual access to the tissue of interest may be hindered, for example, by the accumulation of cell debris on the coverslip. In such cases, the R.MIW can be opened before or immediately after an imaging session to allow cleaning of the entire visible tissue area and lid. The removable lid also allows manipulations of the tissue as well as local application of substances, such as specific inhibitors and therapies, labeling dyes, or specific cell types of interest.
This method overcomes the limitation of the previously described mammary skinflap procedures4,7,8,13, which are limited to one imaging session, and can visualize processes such as branching morphogenesis (Figure 5), homeostatic tissue turnover (Figure 6), or tumor growth at a cellular level (Figure 3B). Yet, at the same time, the R.MIW permits local manipulation of the tissue in between imaging sessions, which comprises a great asset of the R.MIW compared to the previously published MIW3,18 and all other imaging windows20,22. When opening the R.MIW, it is important to maintain aseptic conditions at all times to prevent any source of infection. The ability to open the R.MIW in an aseptic environment allows optimizing the imaging conditions prior to every imaging session, which greatly improves long-term visual access to the tissue of interest. Specifically, in the mammary gland, which is embedded in an adipocyte-rich stroma, this is of great value. Moreover, the replaceable lid could uniquely enable the local administration of therapeutics, different cell types, labeling dyes, image-guided microdissection, or any other local manipulation of the tissue without the need to terminate the IVM experiment. For instance, to study tumor initiation, specific cancer cell populations could be injected directly in the ductal tree or stroma at a determined IVM time-point and precise mammary gland location, as long as this ROI is accessible through the R.MIW ring. To study tumor progression, specific cancer cell populations (at a specific location, in a specific micro-environment, or with a specific behavior) could be photo-marked during the IVM session and subsequently microdissected using a fluorescent dissection microscope after the opening of the R.MIW. Isolated cells could be further processed for downstream analyses, such as (single-cell) mRNA sequencing. By using this approach, one could couple in vivo cell behavior to molecular expression profiles. The advantage of local drug administration enabled by the R.MIW allows the tissue to be imaged prior to and directly after treatment. The interval needed to remove the mouse from the imaging box and to subsequently perform local drug administration after opening the R.MIW lid can be performed in minutes, which allows the immediate phase capture of fast-acting drugs.
We show that IVM of the mammary gland through the R.MIW is compatible with many different fluorescent reporter mouse models. The adipose-rich environment is challenging to image, and therefore the use of bright fluorophores is recommended. However, as shown here, even less bright fluorophores such as mCFP can be visualized through the R.MIW in optimal imaging conditions. Inevitably, the fat pad will preclude the imaging of the deeper ductal structures, and limit the imaging to the more superficial ducts. A low-resolution overview image at the start of each IVM experiment will help to identify the ductal structures of interest that are sufficiently superficial for high-resolution imaging. Local tissue manipulation, removal of connective tissue, or repositioning of the adipose tissue after opening the R.MIW can optimize the IVM for specific ROIs that are overlaid by adipose tissue. This is an important advantage over all previous window designs, which do not allow to perform these manipulations and would require complete removal of the window itself. Specifically, for visualization of the inguinal lymph node, it is recommended to gently remove the overlying fatty tissue, which reduces light scattering and enables high-resolution imaging. When manipulating the tissue, always keep aseptic conditions and prevent bleeding or serious damage to the tissue, as they may affect the processes being studied during the IVM experiment.
The R.MIW is made of titanium, a material that is commonly used in clinical practice to replace hard tissues such as joints or bone plates. Titanium has several advantages over steel windows, including its lightweight and inert character21. Recently, several other materials were used to generate novel imaging window types, including the flexible silicon window20. In contrast to the R.MIW, the flexible window does not require any sutures for implantation and is suitable for nearly any anatomical position, specifically in the case of soft and fragile tissues. Silicon windows have a minimal impact on animal motility due to their lightweight and deformable nature and maybe more suited in experiments studying rapid tissue expansion and growth20. Another advantage over the titanium version is that silicon windows are compatible with other imaging modalities, including magnetic resonance imaging20,27. However, it will be important to keep in mind that objectives are optimized for 0.17 mm glass coverslips. Moreover, mammary tissue is susceptible to breathing movements, which are hard to restrict using the flexible window, especially when using an inverted microscope. Breathing artifacts are minimized by the R.MIW design and the fixation of the R.MIW in the inlay of the imaging box. As a result, images acquired using the proposed R.MIW setup are not distorted due to breathing artifacts. However, minor drifts in tissue localization can occur, which are usually gradual and can be corrected by using post-acquisition motion correction software28. With the increasing toolbox of IVM technologies2,20, the specific requirements for each experiment will eventually determine the best way of in vivo visualization of the tissue of interest. Different window designs have different advantages and disadvantages and depending on the research question, the available microscopy setup, the required spatial and temporal resolution, and the total time span of the studied process, the optimal approach needs to be determined.
In summary, the R.MIW facilitates high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease over multiple days to weeks.

The mammary epithelium is a unique secretory organ present in mammals, which produces and secretes milk to nourish offspring. Throughout life, the mammary gland undergoes multiple rounds of development and growth, which are accompanied by structural and functional changes of the tissue1. Depending on the developmental stage, the cell types contributing to tissue remodeling are different, as well as the location within the ductal tree.
Multiphoton intravital microscopy (IVM) allows the study of mammary cell dynamics in vivo in the native and minimally perturbed setting2,3,4. To obtain visual access to the mammary gland, several temporary ex vivo or skin flap imaging techniques have been published during different stages of mammary gland development, including puberty4,5,6,7, adulthood2,8, lactation9,10,11,12, and tumor dynamics13,14,15. Although these techniques result in high spatially and temporally resolved imaging of mammary cell dynamics, the time frame is limited to hours whereas most mammary gland remodeling processes take days to weeks. Therefore, methods that allow optical access to the tissue of interest for extended time frames are required. Over the years, several permanent imaging windows have been developed for mammary tumor imaging15,16,17,18, including a titanium mammary imaging window (MIW)2,3,19. Although very useful to study mammary tumor growth, visualization of the healthy mammary gland structure remained limited to a few days. Recently, a flexible silicon imaging window was developed, which allows visualization of the pubertal mammary gland over multiple weeks20. However, the mammary gland is embedded in an adipocyte-rich fat pad, which leads to extensive light-scattering and as a result limited visibility of the mammary ductal structures. Therefore, superior imaging conditions are required at all times to visualize tissue dynamics over prolonged periods of time in the mammary gland. Neither the classical MIW, nor the flexible silicon window&#160;allow for tissue manipulation or optimization of physical tissue location before imaging, as the window forms a closed system after surgery and implantation. As a result, optimal optical access to the underlying mammary tissue is likely to become precluded over longer time periods. In contrast, the skin flap technique does allow for optimization and repositioning of the tissue during the imaging session and a skin flap can be repeated multiple times2. However, repeated imaging sessions through a skin flap are only possible when sufficient time (at least 7 days) is allocated in between surgeries to allow recovery of the skin and is therefore mostly suited to study processes at longer time scales. Moreover, it is advisable to not perform this procedure many times because of its invasive nature and large risk of infections and scarring upon wound closure.
To overcome these limitations, i.e., to ensure optimal imaging conditions for a prolonged period of time at a high frequency&#160;and at the same time allow tissue manipulation, an improved titanium version of the MIW with a replaceable lid (R.MIW) was designed to visualize the healthy and diseased mammary gland over multiple days to weeks2 (Figure 1A,B). The R.MIW was custom designed to provide optimal tissue access, enabling direct tissue manipulation over the entire duration of the IVM experiment, and thereby permits visualization of the mammary gland at the right time and place over prolonged periods of time. When closed, the R.MIW forms an airtight system comparable to the classical MIW (Figure 1C). When opened under aseptic conditions, the R.MIW allows local tissue manipulation to improve optical access and also enables local administration of substances, such as pathway inhibitors or agonists, injection of different cell types of interest, such as cancer cell or immune cell populations, or addition of tissue labeling-dyes. The lid can be opened at any moment in between imaging sessions without causing&#160;damage to the underlying tissue.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63326/63326fig01.jpg" /> 
Figure 1: Design of the mammary imaging window with a replaceable lid. (A) Top view and side view of the replaceable lid of the mammary imaging window&#160;with a 10 mm coverglass glued to the ring. (B)&#160;Top view and side view of the mammary imaging window, which consists of an outer ring and inner ring&#160;with a groove in between to secure the window within the skin of the mouse using a purse-string suture. The outer ring has a small groove that fits the four projections (arms) of the lid. (C) Cartoon and pictures demonstrating the opening and closing mechanisms of the lid. The slanting projections make sure the lid is fixed inside the window frame. This figure has been modified from Messal et al. 2. <a href="https://www.jove.com/files/ftp_upload/63326/63326fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This protocol describes the design and implantation procedure of the R.MIW, as well as a longitudinal IVM strategy to revisit the same mammary ducts and their visualization at a cellular resolution. The R.MIW allows following cell divisions and morphological changes during different developmental phases of the mammary gland in diverse fluorescent reporter mouse models. Taken together, the R.MIW facilitates high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>0.9% NaCl</td>
			<td>BD</td>
			<td>306573</td>
			<td>Other brands available</td>
		</tr>
		<tr>
			<td>10 mm round coverglass</td>
			<td>Fisher scientific</td>
			<td>10696365</td>
			<td>Other brands available</td>
		</tr>
		<tr>
			<td>5-0 braided silk suturs</td>
			<td>Ethicon</td>
			<td>W580</td>
			<td></td>
		</tr>
		<tr>
			<td>80% Ethanol</td>
			<td>homemade</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia induction box</td>
			<td>Kentscientific</td>
			<td>VetFlo-MSEKIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine (Vetergesic&#174;)</td>
			<td>Ecuphar</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tips (sterile)</td>
			<td>Fisher scientific</td>
			<td>13113743</td>
			<td>Other brands available</td>
		</tr>
		<tr>
			<td>Cyanoacrylate adhesive</td>
			<td>Loctite</td>
			<td>NA</td>
			<td>Other brands available</td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Duratears</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexible butterfly needle</td>
			<td>Greiner Bio-One</td>
			<td>450120</td>
			<td>Other brands available</td>
		</tr>
		<tr>
			<td>Graefe forceps (blunt)</td>
			<td>Fine Science Tools</td>
			<td>11051-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Kentscientific</td>
			<td>RightTemp Jr.</td>
			<td>Other brands available</td>
		</tr>
		<tr>
			<td>Imaging box</td>
			<td>Custom made</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Infuse nutrient mixture</td>
			<td>Braun</td>
			<td>Nutriflex special 70/240</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td>BD</td>
			<td>324911</td>
			<td>Other brands available</td>
		</tr>
		<tr>
			<td>Inverted multi-photon microscope with automated stage</td>
			<td>Leica Microsystems</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer</td>
			<td>Kentscientific</td>
			<td>VetFlo-1231K</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Fine Science Tools</td>
			<td>12510-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma-Aldrich</td>
			<td>P7793</td>
			<td>semi-transparent tape</td>
		</tr>
		<tr>
			<td>Paper tape tesa</td>
			<td>Tesa</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Petroleum Jelly</td>
			<td>Vaseline</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Fisher scientific</td>
			<td>11904325</td>
			<td>Other brands available</td>
		</tr>
		<tr>
			<td>Silicon tubing</td>
			<td>Solutions Elastomeres</td>
			<td>NA</td>
			<td>Any houseware</td>
		</tr>
		<tr>
			<td>Spring scissors (small)</td>
			<td>Fine Science Tools</td>
			<td>15018-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile PBS</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (10 ml)</td>
			<td>BD</td>
			<td>305482</td>
			<td>Other brands available</td>
		</tr>
		<tr>
			<td>Thin forceps</td>
			<td>Fine Science Tools</td>
			<td>11413-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium lid for mammary imaging window</td>
			<td>Custom made</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium mammary imaging window</td>
			<td>Custom made</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Wooden sticks toothpicks</td>
			<td>Fisher scientific</td>
			<td>NC1678836</td>
			<td>Other brands available</td>
		</tr>
	</tbody>
</table>

longitudinal intravital microscopy using a mammary imaging window with replaceable lid

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

To study the proliferative dynamics of mammary epithelial cells during the different developmental stages of the mammary gland the described protocol was performed. The design of the R.MIW is depicted in Figure 1, and the procedure to implant an R.MIW is summarized in Figure 2. The R.MIW is surgically implanted between the mammary gland and the skin. A suture is used to hold within the window ring and prevent the mice from pulling on the sutures (Figure 2). The sutures holding the R.MIW are made of non-absorbable silk, which has hypoallergenic properties, a soft texture, and is easy to handle. The R.MIW ring is made of titanium, one of the most biocompatible materials that do not promote inflammation or necrosis after implantation21. If the aseptic conditions are followed and the sutures are well executed, the R.MIW mammary gland implantation poses a low risk of postoperative complications. Moreover, unlike abdominal imaging window implantation22, R.MIW implantation is not a limiting factor for mouse survival because the mammary gland is not a vital organ and allows daily imaging up to the limit specified in the ethical protocol. The only factor that limits the duration of window implantation is the homeostatic turnover of the skin, which will eventually lead to the sutures falling out after 4 - 6 weeks. Therefore, it is important to regularly inspect the stability of the sutures. If desired, the sutures can be removed and replaced by a new purse-string suture under aseptic conditions (as described in steps 3.5 - 3.8) to reassure window stability.
A major challenge when using the multiday imaging approach is retracing an ROI on consecutive days. To this end, a quick overview scan can be included before selecting the ROI(s) (Figure 3B). Multiple tissue landmarks can be used to retrace the same region of the tissue, including the collagen network signal (visualized by second harmonic&#160;generation), tissue structure, as well as local patterns of cells differentially or stochastically labeled with dyes or fluorescent proteins (Figure 3B). In this representative example, an R.MIW was implanted onto the 4th&#160;mammary gland of&#160;an MMTV-PyMT;R26-CreERT2het;R26-Confettihet female mouse at the onset of palpable tumor formation.&#160;Subsequently, stochastic recombination was induced by injection of 1.5 mg tamoxifen, resulting in recombination of the Confetti construct in some cells (Figure 3C). The recombined tumor cells were followed for a period of 20 days (Figure 3B,C). If imaging of the same region of the mammary gland over consecutive days is precluded, the window can be opened in an aseptic environment (as described in step 5.1) to further clear up, and reposition the tissue to improve visibility and image acquisition (Figure 3A).
Using this method, the developing mammary gland during puberty was followed at the single-cell level. Repeated IVM through an R.MIW was performed using R26-CreERT2het;R26-mTmGhet&#160;female mice between 4-6 weeks of age, in which all cells are labeled with membrane tdTomato (Figure 4A)23. Mice were injected with a low dose of tamoxifen (0.2 mg/25 g body weight), resulting in sporadic recombination of the mTmG construct, causing a change from red to green in some cells in all tissues, including the mammary gland (Figure 4B). The same ROIs were revisited over multiple days to visualize morphological changes within the mammary gland, including ductal elongation and ductal branching (Figure 4C) at a cellular resolution. Importantly, this combination of stochastic cell labeling and multi-day IVM also allows visualizing the dynamic changes of the ductal environment, such as the dynamics of single cells in the stroma surrounding the elongating and branching ducts (Figure 4D) as well as the cellular dynamics within the inguinal lymph node (Figure 4E).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63326/63326fig04.jpg" /> 
Figure 4: Multi-day IVM of pubertal branching morphogenesis. (A) Pubertal R26-CreERT2; R26-mTmG mice in age between 4-6 weeks were injected with a low dose of tamoxifen leading to Cre-mediated recombination of the R26-mTmG allele and were imaged on days 1, 3, and 5 to follow the dynamic changes in developing mammary gland. (B) Schematic representation of the R26-mTmG mouse&#160;construct, which in non-recombined conditions results in ubiquitous expression of membrane tdTomato (mT). Upon Cre-mediated recombination, mT is switched for membrane eGFP (mG) expression. (C)&#160;3D rendering of an elongating and branching mammary duct over multiple days imaged through an R.MIW in an R26-CreERT2; R26-mTmG female mouse at 6 weeks of age. (D)&#160;Single Z-plane images of the branching tip (top panels) and the elongating branch (bottom panels). mT is depicted in red, and mG in cyan, scale bars represent 100 &#181;m (A and B). Single cells in the stroma are highlighted by white asterisks. Note that signal intensity was manually increased to highlight the single cells in the stroma, leading to a slightly overexposed appearance of the mammary epithelial cells. (E)&#160;Representative images of the inguinal lymph node of an R26-CreERT2; R26-mTmG female mouse imaged through an R.MIW, showing an overview tile scan (left panel), zoom images (right panels) of a single Z-plane (top), and a 3D rendering (bottom). Second harmonic generation (collagen I) is shown in green, mT in red, and mG in cyan. Scale bars represent 500 &#181;m (overview tilescan) and 100 &#181;m (zoom images). Figure panels C and D are modified from Messal et al. 2. <a href="https://www.jove.com/files/ftp_upload/63326/63326fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Similarly, in the adult mammary gland, the proposed R.MIW approach allows visualization of the same ductal structures over multiple days with uncompromised visibility. Even the use of less bright fluorescent reporter models24, such as the Cdh1-mCFP (Ecadherin-mCFP) mouse model, permits visualization of ductal stability and subtle morphological changes at a cellular resolution (Figure 5). Note that visibility between day 3 and day 5 improved significantly after tissue repositioning (Figure 5).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63326/63326fig05.jpg" /> 
Figure 5: Multi-day imaging of the adult mammary gland. 3D rendered images of a mammary ductal structure of an adult female Cdh1-mCFP (cyan, marking the Ecadherin-positive luminal cells expression) mouse over multiple consecutive days. The collagen I pattern (second harmonic generation, magenta) was used to retrace the same ROI, and the Cdh1-mCFP signal was used to mark the ductal (luminal) cells. Note that visibility after day 3 was improved by the opening of the R.MIW lid and repositioning of the tissue. Scale bars represent 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63326/63326fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To assess proliferative heterogeneity of the mammary epithelial cells during the hormonal cycle at the single-cell level, the photo-convertible Kikume Green-Red (KikGR) reporter mouse model25,26 was used, which has ubiquitous expression of the KikGR protein. KikGR is a bright fluorophore that undergoes green-to-red conversion upon exposure to violet light and the red/green ratio can be used as a proxy for the proliferative activity of a cell2 (Figure 6A,B). Using the R.MIW approach, we followed the same cells within the ductal tree of the adult mammary gland over several days and found previously unanticipated proliferative heterogeneity throughout the ductal tree (Figure 6C). High and low proliferative cells were equally distributed over the different converted areas (Figure 6C,D). Strikingly, at the local level, neighboring cells showed large differences in their red/green ratio (Figure 6C,D). Quantification of the red/green ratio of different cells (as a proxy for their proliferative activity) 10 days after photo-conversion, revealed highly variable dilution rates of some cells within the same ductal micro-environment (Figure 6E). Together, these data reveal a remarkable local proliferative heterogeneity within the adult mammary gland, and at the same time, a global uniform turnover rate.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63326/63326fig06.jpg" /> 
Figure 6: Longitudinal IVM of proliferative heterogeneity in the adult mammary gland using the KikGR mouse model. (A)&#160;KikGR mice were imaged at day 0 before and after exposure to violet light. The imaging sessions were repeated on days 2, 6, and 10 after conversion. (B) Schematic depiction of the hypothetical outcomes upon photo-conversion of KikGR mammary epithelial cells following proliferation (left panel) and no proliferation (right panel) as a function of time. (C)&#160;The same area of the mammary gland was imaged through an R.MIW over a period of 10 days. Smaller regions were photo-converted on day 0 and show a similar dilution rate of the Kikume red signal over time, indicating an equal turnover rate throughout the epithelium. (D)&#160;Zoom images (single Z-planes) of the indicated regions in panel C show proliferative heterogeneity at the cellular level 6 and 10 days after photo-conversion. Scale bars represent 100 &#181;m (panel C) and 10 &#181;m (panel D). (E)&#160;Quantification of the red/green ratio of randomly selected cells (n = 48 cells) in three photo-converted areas. A high red/green ratio is indicative of a low dilution rate and low-proliferative activity, whereas a low red/green ratio is indicative of a high dilution rate and proliferation. Figure panels C- E have been modified from Messal et al. 2. <a href="https://www.jove.com/files/ftp_upload/63326/63326fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid at JoVE.com

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

This protocol describes a novel mammary imaging window with a replaceable lid (R.MIW). Intravital microscopy after implantation of the R.MIW allows for longitudinal and multi-day imaging of the healthy and diseased mammary gland with a cellular resolution during the different developmental stages.

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in ...

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