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Biology

Intravital Subcellular Microscopy of the Mammary Gland

Published: June 24, 2022 doi: 10.3791/63674

Abstract

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676 or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs ≥ 0.7 µm. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 µm or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

Introduction

Intravital microscopy of the mouse mammary gland is attracting increased attention as a powerful method for analyzing a whole range of biological phenomena, including the origin and differentiation of stem cells1,2, the progression of metastatic tumors3,4,5, and the role of ductal macrophages throughout mammary development and involution6. Through the development of Intravital Subcellular Microscopy (ISMic)7, investigations have been extended to membrane traffic and secretory mechanisms during lactation8,9, and oxytocin-mediated contraction of myoepithelial cells9,10. Two main methods have been developed that take advantage of the gland's accessibility between the skin and body wall.

In the first approach, an acrylic or glass window is inserted into the skin and secured with a metal retaining ring1,3,11. The mice tolerate the surgery well, and various phenomena can be analyzed on an intermittent basis in the same animal over several weeks. This method has proved especially useful for lineage tracing1,12 and monitoring the invasion and progression of mammary tumors in situ3,11. However, resolution below the whole-cell level has proven difficult because the gland is still attached to the body wall and is thus subject to motion artifacts caused by respiration and heartbeat.

In the second approach, the gland is surgically exposed on a skin flap with intact vasculature and stabilized on the microscope stage with spacers4,9,13. A portion of the gland is thus effectively separated from the abdominal wall, and motion artifacts are minimized. In most cases, the exposed parenchyma is placed directly on the coverslip with the mouse ventral side down on an inverted microscope. In a recent modification, the mouse was placed supine on an upright microscope, and the exposed gland was protected in a fluid-filled cell sealed with a coverslip2. This latter configuration allows access to the parenchymal surface for experimental manipulation during imaging. Resolution down to <1 µm, in either case, permits analysis of intracellular processes, as exemplified by the tracking of lipid droplets (LDs) in mammary epithelial cells9.

The present protocol details a facile method for the intravital imaging of mammary epithelial cells at the sub-cellular level using the biogenesis, transport, and secretion of LDs during lactation as an example. This approach is widely applicable to many other processes, including the transport and secretion of milk proteins14, the transcytosis of proteins from the serosal side of the epithelium to the alveolar lumen15,16, and the remodeling of the gland during involution17,18.

Mice expressing a fluorescent protein are preferred for most intravital experiments to facilitate the selection of appropriate areas for imaging and as a morphological reference marker. A wide range of suitable transgenic and knock-in mice are available, which express fluorescent protein markers in cellular compartments, cytoskeletal elements, membranes, and organelles19. In the examples given, the EGFPcyto FvB mouse was used, in which enhanced green fluorescent protein (EGFP) is targeted to the cytoplasm in most cells20 (denoted EGFPcyto), and the C57BL/6J Tomato (mT/mG) mouse21, which is a double fluorescent Cre line encoding tdTomato and EGFP genes. EGFP expression is enabled through Cre-mediated excision of the tdTomato gene. Either fluorophore is targeted to the plasma membrane in most cells through a sequon derived from the MARCKS protein21. In this work, mice expressing the red tdTomato fluorophore are denoted tdTomatomembr (mT), and mice expressing EGFP, after excision of the tdTomato gene are denoted EGFPmembr (mG).

Mice have five pairs of mammary glands on either side of the ventral midline, three in the thoracic region (numbered 1-3) and two in the inguinal region (numbered 4-5) (Figure 1A). For ISMic, the inguinal glands are the most accessible and easiest to stabilize, as they are furthest away from global motions associated with respiration and heartbeat in the thorax.

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Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee of the Center for Cancer Research, National Cancer Institute, the National Institutes of Health in compliance with the US National Research Council's Guide for the Care and Use of Laboratory Animals, the US Public Health Service's Policy on Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals. For this work, the number 4 glands of female primiparous mice (aged 4-5 months, day 10 of lactation) were surgically prepared in the right-hand supine position (Figure 1A).

1. Animal preparation

  1. Weigh the mouse on a top-loading balance to calculate the amount of anesthetic required.
  2. During and after applying anesthetics, maintain body temperature by placing the mouse on a warming pad.
  3. Anesthetize the mouse by brief exposure to isoflurane (3.0%-3.5%) in an oxygen-saturated respiration chamber (see Table of Materials) for 1-2 min, followed by an intraperitoneal injection of a mixture of 100 mg/kg of ketamine and 10 mg/kg of xylazine.
  4. Maintain animals under a deep plane of anesthesia during both surgery and imaging by further applying titrated doses of xylazine and ketamine (half to a quarter of the initial dose outlined in step 1.3) through an indwelling catheter (see step 3.4) every 45 min or so, as required.
    NOTE: An optimal plane of anesthesia is most easily maintained by checking for palpebral and toe pinch reflexes every 10-20 min. The temperature of conscious mice is 36.5-38 °C, which drops to 34.5 °C under anesthesia.
  5. Cross-foster the pups22 if donor mice at a similar stage of lactation are available; otherwise, euthanize the litter.
    ​NOTE: Pups <10 days of age should be euthanized by exposure to 3.0% isopentane followed by decapitation. Pups ≥10 days of age should be immersed in a tank of CO2, followed by cervical dislocation.

2. Surgery procedure

  1. Keep the mouse warm throughout surgery on a warming pad. Shave the fur from the skin surrounding the number 4 and 5 mammary glands (supine, right side) using a hand-held electric razor. Clean the shaved area with three alternating betadine and alcohol scrubs.
  2. Wipe all surgical instruments with 70% alcohol. Make a mid-line incision of ~1 cm close to the fourth nipple by pinching the skin with tweezers and cutting the raised skin with sharp scissors. Make circular incisions around the gland in both cranial and caudal directions and carefully peel the skin away from the abdomen. Keep the exposed skin moist with physiological saline.
  3. To minimize blood loss, seal any prominent blood vessels with a hand-held cauterizer (see Table of Materials) before cutting through them. Any exogenous blood should be promptly removed with physiological saline to avoid subsequent loss of optical resolution.
  4. Remove superficial connective and adipose tissue by gently teasing the surface layer with fine forceps.
  5. Keep the exposed gland moist with physiological saline and protect the abdominal wall with gel and semi-transparent, flexible, thermoplastic film (see Table of Materials).
    NOTE: Steps 2.2-2.5 create a flap of skin with a portion of the gland and associated vasculature separated from the abdominal wall (Figure 1Bi-iv).
  6. If required, treat the gland with exogenous agents, e.g., pharmacological agents or organelle-specific dyes, by bathing the exposed gland during surgery.
    ​NOTE: In the examples given, LDs in either an EGFPcyto or EGFPmembr (mG) mouse were labeled with 1.0 mL of boron-dipyrromethene (BODIPY)665/676 (10 mM stock solution in dimethyl sulphoxide diluted 1 to a 1,000-fold in saline and applied for <1 h) and in the tdTomatomembr (mT) or EGFPmembr (mG) mouse, with 0.5 mL of monodansylpentane23 (0.1M stock solution in dimethyl sulphoxide diluted 1 to a 1,000-fold in saline and applied for 5 min).

3. Imaging preparation

  1. Carefully position the mouse with the abdominal side down on a heated (37 °C) inverted microscope stage, such that the skin flap extends onto the central cover glass (30 mm) (Figure 1Biv). Protect the exposed areas with a thin layer of gel.
  2. Stabilize the skin flap with custom-made spacers to cushion the gland from motion artifacts caused by breathing and heartbeat. To achieve this, place a notched spacer made from three taped cotton sticks (Figure 1Ci) between the skin flap and the body wall and tape the rear leg and tail to the stage behind the spacer (Figure 1Bv).
  3. Prevent the skin flap from sliding during imaging by securely taping a plastic cover (Figure 1Cii) at either end on the stage opening (Figure 1Bv).
  4. Insert a subcutaneous indwelling catheter under the dorsal skin attached to a tube, syringe, and pump (see Table of Materials).
  5. Confirm that the skin flap is stable with adequate blood flow by conventional fluorescence microscopy (Figure 1Bvi).
  6. Cover the mouse with sponge gauze (see Table of Materials) to keep warm during imaging,
    ​NOTE: Steps 3.1-3.5 are the most critical in ensuring the acquisition of high-resolution videos suitable for quantitative analysis. There is no point in proceeding beyond this stage unless tissue stability has been confirmed.

4. Microscopy

  1. For conventional confocal microscopy of the EGFPcyto or EGFPmembr (mG) mouse labeled with BODIPY665/676 (Figure 2, Video 1, and Video 2), use an inverted microscope equipped with a preheated 60x oil immersion objective, maintained at 37 °C (see Table of Materials).
    1. Separately detect EGFP and BODIPY665/676 using 488 and 633 lasers, respectively; for EGFP, excitation 488 nm, and emission 560 nm, with band-pass filter BA 505-605; for BODIPY665/676, excitation 633 nm and emission 668 nm, with band-pass filter BA 655-755.
    2. Collect images by line scan at either 4 or 8 µs/pixel (512 x 512 pixels; 12 bits per pixel) every 5 s or 10 s for 1-2 h and store as TIFF files. Manually maintain scanned areas in-frame by correcting for x/y drift throughout the imaging cycle. Construct 3-D images from z-scans using software associated with the microscope (see Table of Materials).
  2. For two-photon microscopy of the EGFPmembr (mG) mouse labeled with monodansylpentane (Figure 3, Video 3), use an inverted microscope equipped with a tunable laser and a 37 °C preheated 30x objective (see Table of Materials).
    1. Excite the gland at 910 nm and detect monodansylpentane and EGFP with two GaAs detectors, using 410-460 nm and 495-540 nm band-pass filters for blue and green emissions, respectively.
    2. Collect images by line scan at 2 µs/pixel (320 x 320 pixels; 16 bits per pixel) and store them as OIR files. Manually maintain scanned areas in-frame by correcting x/y drift throughout the imaging cycle. Construct 3-D images from z-scans using software associated with the microscope.
  3. For two-photon microscopy of the tdTomatomembr (mT) mouse labeled with monodansylpentane (Figure 4, Video 4), use an inverted microscope with tunable lasers, and a 37 °C preheated 63x objective (see Table of Materials).
    1. Excite specimens at 800 nm and 1,060 nm simultaneously and detect monodansylpentane with a HyD hybrid detector set at emission 418-471 nm and TdTomato with another HyD hybrid detector set at emission 595-667 nm.
    2. Collect the images by line scan with four lines averaging at 200 Hz (512 x 512 pixels; 12 bits per pixel) and store them as LIF files. Manually maintain scanned areas in-frame by correcting x/y drift throughout the imaging cycle. Construct 3-D images from z scans using software associated with the microscope.

5. Euthanasia

  1. Euthanize the mice at the end of imaging by immersing them in a tank of CO2 following institutionally approved protocol.

6. Creation of real-time videos

  1. Convert time sequences into TIFF files, and then into videos using appropriate software (see Table of Materials).
    NOTE: Quantitative analysis of specific videos is beyond the scope of this paper and will require specific methods for the solution of specific problems. For example, see Ebrahim et al.24 for the role of actinomyosin complexes in exocytosis of saliva from the salivary gland, Meyer et al.25 for the dynamics of bile secretion from the liver, and Masedunskas et al.9 for analysis of LD secretion from mammary epithelial cells.

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Representative Results

Milk is secreted from polarized alveolar epithelial cells, which differentiate during pregnancy from the buds of an extensive ductular tree26 (Figure 2A). Precursors for milk synthesis are assimilated across basal/lateral membranes and completed products are secreted across the apical surface into a central "milk space". The basal side of each alveolus is covered by a stellate array of myoepithelial cells (Figure 2A), which are protected on the serosal side by a basement membrane (not shown in Figure 2A). Oxytocin-induced contractions of the myoepithelium at "milk let-down" release LDs from the secretory cells9,27 and force completed milk products through the ductular tree to the nipple and suckling neonate10. Gland physiology is maintained throughout by a constant supply of blood-borne constituents from an underlying capillary bed.

Using ISMic, imaging is possible to a depth of 15-20 µm through the capillary bed, extracellular matrix, myoepithelial and secretory cells into proximal luminal spaces (Figure 2B). In the case of the EGFPcyto mouse (Figure 2Bi-iv), the cytoplasm of secretory epithelial cells was distinctly labeled by EGFP compared with the less fluorescent capillary bed and myoepithelium. LDs stained with BODIPY665/676 appeared as round fluorescent bodies of varying sizes and were especially prominent toward the apical surface. In the EGFPmembr (mG) mouse, EGFP highlighted the plasma membranes of capillary and secretory epithelial cells, and BODIPY-stained LDs were obvious throughout the cytoplasm (Figure 2Bv-viii).

Intravital microscopy of the EGFPcyto mouse highlighted EGFP-labeled epithelial cells and resident macrophages6,9, which appeared as dark unlabeled cells in intimate and dynamic association with the epithelium. Close inspection of the epithelial cells revealed numerous unstained particles streaming toward apical surfaces (Video 1). At higher power, time-lapse analysis of BODIPY665/676 stained glands showed that a fraction of these particles were labeled LDs (Video 2). LDs moved from basal regions to the apical cytoplasm at highly variable speeds (0.02-4.7 µm/min) along potential tracks. ~50% of these droplets displayed directed (superdiffusive) motion28, possibly indicating transport on elements of the cytoskeleton9. LDs fused with each other in transit (Video 2), especially in apical nucleation centers associated with the plasma membrane. LDs even increased in volume as they were budding from the cell. Final release into the central milk space required oxytocin-mediated contraction of the myoepithelium. Thus showing for the first time that droplets ≥0.7 µm in diameter are secreted in a regulated oxytocin-dependent manner9.

By two-photon microscopy, imaging to a depth of at least 60 µm is possible (Video 3 and Video 4). To label LDs, the far-red dye BODIPY665/676 was replaced with the blue fluorophore monodansylpentane23 because of emission overlap with tdTomato. With an absorbance peak below 400 nm and an emission maximum of 420-480 nm, monodansylpentane was fully compatible with both green and red fluorophores (EGFP, Video 3; tdTomato, Video 4). In addition, it was remarkably stable for at least 1 h, which is an essential attribute for tracking the comparatively slow movement and accretion of LDs in mammary cells (Video 2)9.

Two-photon microscopy of the mammary glands of either tdTomatomembr (mT) or EGFPmembr (mG) mice showed similar morphological features as single-photon confocal microscopy (Figure 2B) but to a depth, which extended well into core luminal spaces (Video 3 and Video 4; Figure 3 and Figure 4). The red tdTomato fluorophore was especially useful for labeling the capillary endothelium (Video 4, Figure 4A), unlike EGFP in the EGFPcyto mouse (Figure 2Bi). Two-photon microscopy allows a more comprehensive, in-depth survey of gland morphology than single-photon confocal microscopy. However, because of resolution issues, it is less useful for ISMic.

Figure 1
Figure 1: Preparation of skin flap and spacers used for stabilization. (A) Numerical classification of mouse mammary glands and position of skin flap used for imaging. (B) Preparation of mammary gland (number 4) for intravital imaging. (i-iii) Surgical preparation of the skin flap having the mammary gland attached and with associated vasculature. (iv) Positioning of the exposed gland on the microscope stage, (v) Final position of the animal for imaging, (vi) Coventional fluorescence microscopy to check gland for stability and blood flow. (C) Spacers used to stabilize skin flap. (i) Spacer was created to separate the body and rear leg from the skin flap made by taping three cotton sticks together. The central section of one stick was removed to create a notch to accommodate the skin flap. (ii) One example of a plastic piece used to cover and stabilize the skin flap. A range of different sizes is helpful for various sized glands. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Confocal intravital microscopy of the lactating mouse mammary gland. (A) Line drawing of a mammary alveolus and associated duct. The mammary epithelium (dark gray) is surrounded by a capillary bed and myoepithelial cells (light gray), which enwrap the entire alveolus. Milk is secreted into the central lumen and is expelled into the ducts during the oxytocin-mediated contraction of the myoepithelium. (B) In situ BODIPY665/676-labeled intravital confocal sections from (i-iv), the EGFPcyto mouse, showing the (i) basal, (ii-iii) medial, and (iv) apical regions of the secretory epithelium. In comparison, the myoepithelium and capillary endothelial cells are relatively dark because they express lower amounts of EGFP; (v-viii) the EGFPmembr (mG) mouse, showing (v) basal, (vi-vii) medial, and (viii) apical regions of the secretory epithelium with the targeting of EGFP to apical, lateral, and basal plasma membranes. The dotted line in vii encloses a grazing section of the cell apex with accumulating LDs. BODIPY-stained LDs (magenta); EGFP (green); cap, capillary; apm, apical plasma membrane; myo, myoepithelial cell; ld, lipid droplet; n, nucleus. Scale bars, 20 µm. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Selected optical slices of the lactating mammary gland of an EGFPmembr (mG) mouse by two-photon microscopy. (A) Collagen fibers (arrowheads) at the surface (2 µm) are visualized in the monodansylpentane channel through second-harmonic generation (SHG). (B) Further into the parenchyma at 8 µm, the secretory epithelium is revealed with basal, lateral, and apical plasma membranes (green) and monodansylpentane-stained LDs (blue). A grazing section of the apical surface (within the white line) reveals LDs accumulating at the cell apex, adjacent to an area just below the apical surface, in which there are lateral connections between neighboring cells in the epithelium (arrows). (C) The central lumen becomes obvious further into the alveolus at 16 µm. LDs, which appear to have been released into the lumen, are still associated with the apical plasma membranes of cells further into the tissue27. EGFP (green), monodansylpentane-stained LDs (blue). Scale bars, 20 µm. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Selected optical slices of the lactating mammary gland of a tdTomatomembr (mT) mouse by two-photon microscopy. (A) The capillary bed is highlighted close to the surface (arrowheads), together with basal views of the secretory epithelium (arrows). (B) Further into the alveoli at 16 µm, capillaries appear in cross-sections (arrowheads), and monodansylpentane-stained LDs (blue) accumulate in apical regions seen in the cross-section. (C) At 34 µm, the extent of luminal spaces becomes obvious. tdTomato (red), monodansylpentane-stained LDs (blue). Scale bars, 30 µm. Please click here to view a larger version of this figure.

Video 1: Confocal microscopy of the lactating mammary gland of an EGFPcyto mouse. Low power view of an alveolus showing macrophages (motile dark cells) interacting and probing the secretory epithelium. Close inspection of individual epithelial cells reveals unlabeled particles streaming toward the apical surface. That a fraction of these particles are LDs was shown by staining the gland with the hydrophobic dye, BODIPY665/676, during surgery (Video 2). Speed 25 frames/sec for 60 min. Bar 20 µm. Please click here to download this Video.

Video 2: Transit and accretion of LDs in the lactating mammary gland of an EGFPcyto mouse. BODIPY665/676-labeled LDs (white) are transported at variable speeds from basal regions of the epithelium toward the apical surface over approximately 20 min. Some LDs fuse in transit and feed into large LDs at the cell apex and into some that are protruding from the cell. Left side, both EGFP (green) and BODIPY665/676 (white) channels, Right side, BODIPY665/676 channel only. Speed 40 frames/sec for 1 h 19min. Bar 20 µm. Please click here to download this Video.

Video 3: Lactating mammary gland of an EGFPmembr (mG) mouse examined by two-photon microscopy. LDs were stained with monodansylpentane. Channels were imaged simultaneously while scanning in z-direction in 2 µm increments from the surface to a depth of 28 µm (see Figure 3 for details of three selected optical slices). EGFP (green), monodansylpentane-stained LDs (blue). Scale bar, 20 µm. Please click here to download this Video.

Video 4: Lactating mammary gland of a tdTomatomembr (mT) mouse examined by two-photon microscopy. LDs were stained with monodansylpentane. Channels were imaged simultaneously while scanning in z-direction in 2 µm increments to a depth of 62 µm (see Figure 4 for details of three selected optical slices). tdTomato (red), monodansylpentane-stained LDs (blue). Scale bar, 30 µm. Please click here to download this Video.

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Discussion

Whether to use a one- or multiphoton microscope depends upon the questions being asked, the nature and location of the tissue to be imaged, and the resolution required. Multiphoton microscopes are based on generating two or more low-energy photons in the near-infrared, which can penetrate tissues to a greater depth with less phototoxicity than one-photon microscopes29,30. In addition, the fluorophore is only excited at the focal point, which reduces light scattering from the surrounding tissue. Therefore, multiphoton microscopy has become a standard method for studies within the whole mammary gland of cell lineage tracing1,2,12, the progression of tumor metastases3,13, and multicellular interactions2. At low power, two-photon microscopy allows an in-depth global survey of alveoli in the lactating gland and the temporal secretory status of each. However, the resolution of multiphoton microscopes is lower than that possible with single-photon confocal microscopes31, which even in vivo, can approach the diffraction limit. Spinning disk confocal microscopy was recently used to resolve actin and non-muscle-myosin II lattices around secretory granules down to the level of ~0.3 µm in the live mouse salivary gland24. Given the ready accessibility of the mammary epithelium from the capillary bed to the alveolar lumen (Figure 2B), the intravital imaging of many subcellular activities (ISMic), including secretion, endocytosis, and organellar interactions, are all feasible with the application of single-photon confocal microscopy. Close to super-resolution conditions may be achieved using high-power objectives (e.g., 63x), appropriate exogenous and endogenous fluorescent probes and spinning-disk capability. Many secretory activities can be analyzed within relatively short time periods (≤1 h), thus avoiding long-term problems of phototoxicity.

Intravital imaging can become a routine procedure with practice, although initially, it may seem intimidating and require specialized skills. Maintenance of a deep plane of anesthesia is essential throughout surgery and imaging. Under ideal conditions, this is administered by continuous exposure to isoflurane (initially at 3.0%-4.0%, dialed back to 1.0%-1.5% during surgery and imaging)2,32,33. Imaging times of at least 12 h are achievable under such conditions2,4,33. Unfortunately, the cramped facilities common in many microscope facilities precludes reliable removal of excess hazardous isoflurane from the atmosphere. Therefore, in the examples given, ketamine/xylazine mixtures were administered by intraperitoneal injection during surgery and, after that, via an indwelling catheter during imaging. Care should be taken to maintain body temperature throughout the whole process.

Several issues require special attention during surgery, the most challenging being limitation of vasculature damage during exposure of the skin flap. Blood to the inguinal mammary glands (numbers 4 and 5) is supplied by the external pudendal artery, which in the gland splits into cranial and caudal branches34. Following the initial mid-line cut, the cranial artery is easily avoided by cutting around the outer edge of the gland. The caudal incision is more challenging because it requires cutting through the vascular bed between the number 4 and 5 glands. With practice, blood loss can be kept to a minimum by avoiding and, if necessary, cauterizing the major vessels supplying the number 5 gland.

For one- and two-photon confocal microscopy, removing as much connective and adipose tissue as possible is desirable, as adipocytes are responsible for much of the autofluorescence typical of mammary tissue2. This is less of a problem for one-photon confocal microscopy of lactating glands, as the adipose compartment is at a minimum, compared with other stages of the mammary cycle, e.g., in pre-pubertal glands, during early pregnancy and late involution. Provided surface connective tissue is removed, the lactating gland can be imaged through the initial layer of myoepithelial and epithelial cells into luminal spaces with minimal light-scattering issues. Extraneous collagen, adipocytes, and extracellular matrix can be removed with fine tweezers from the surface of the skin flap, taking care to cover exposed surfaces with physiological saline or a gel. For two-photon microscopy of mammary tissue at all stages of development, Dawson et al.2 described surgical procedures for removing connective and adipose tissue from skin-flap preparations in the supine position using an upright microscope.

The most critical step for intravital high-resolution confocal microscopy is stabilizing the skin flap on the microscope stage while maintaining optimal blood flow. This is best achieved by separating the skin flap from the back leg and body with a notched physical barrier (Figure 1Ci), with the leg and tail securely taped down behind the barrier. A rigid plastic cover needs to be taped down on top of the flap to stabilize the tissue without any adverse effect on blood flow. The plastic cover can be further secured by overlaying it with an additional spacer if necessary. This requires trial and error for each preparation using a standard fluorescence microscope to check for adequate blood flow and tissue stability. This same microscope can then be used to select an area of the flap for imaging. Typically, the most stable regions are furthest away from the barrier and body wall. Low power global scanning of the entire skin flap is possible with some microscopes.

Over time, imaging in either 2- or 3-D at a sub-cellular resolution will inevitably lead to drift in the pre-selected area. Drift in x/y is most easily corrected manually throughout the imaging period using points of reference selected at time 0 min. Drift in z is more problematic and is best corrected after imaging has been completed35. At sub-cellular resolution, imaging times of 1-2 h should be adequate for analyzing many aspects of lipid accretion and transport, membrane traffic, and organellar interactions; LD transit from basal regions9 to the cell apex takes ~20 min (Video 2), synthesis, processing and passage of milk proteins through the classical secretory pathway36 takes 30-60 min37 and basal to apical transcytosis of serum albumin and sIgA15,38, less than 1 h. Stable videos can be made for at least 2 h, which should give ample time to analyze many of these activities. At the resolution of whole cells, imaging times over 12 h are feasible4. Unfortunately, because of the surgery required to make the skin flap, this procedure is terminal in most investigators' hands, although using aseptic techniques, Messal et al.39 have recently been able to image the same animals repeatedly for up to 6 months.

The ability to analyze sub-cellular activities in real-time within the mammary epithelium constitutes a groundbreaking advance in the field of mammary gland biology. The current understanding of mammary physiology is based on studies with whole animals, tissue explants, microscopy of fixed material, and analysis of cell lines and tissue homogenates. By intravital imaging, existing paradigms can be reassessed, with the potential for novel discoveries, using kinetic and mechanistic approaches in live animals. In one recent example, the secretion of LDs, long assumed to be a constitutive process, was shown to be regulated through oxytocin-mediated contraction of the myoepithelium9. Progress in this field should be rapid, given the availability of many transgenic mouse lines, which express fluorescent fusion proteins19, the ease of obtaining the secretion by simple milking procedures40, and the accessibility of the gland to experimental manipulation by infusion of pharmacological agents and adenoviral vectors through the nipple41.

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Disclosures

None of the authors have any conflicting interests to declare.

Acknowledgments

The authors thank Sherry Rausch and Samri Gebre (National Cancer Institute, NIH) for animal management and care and James Mather for help in producing a range of plastic spacers. This research was supported [in part] by the Intramural Research Program of the NIH.

Materials

Name Company Catalog Number Comments
488 laser Melles-Griot - CW  laser 50 mW
60x PLAPON oil immersion objective (NA 1.42) Olympus 1-U2B933 Lens Confocal microscope
633 laser Melles-Griot - CW He-Ne laser 12 mW
63x objective (NA 1.40, HC PL APO CS2) Leica 11506350 Lens Two-photon microscope
BA 410-460 nm Chroma - Band-pass filter
BA 495-540 nm Chroma - Band-pass filter
BA 505-605 nm Chroma - Band-pass filter
BA 655-755 nm Chroma - Band-pass filter
Boron-dipyrromethane (BODIPY) 665/676 Thermo Fisher Scientific B3932 Lipid peroxiation sensor
Carbomer-940 Snowdrift Farm 739601480651 Gel
Catheter Terumo SV27EL Winged infusion sets 
Cauterizer  Braintree Scientific, Inc GEM 5917 Cautery system
CMV-Cre mouse  Jackson lab 006054 Mouse line
Coverslip Bioptechs - 30mm diameter coverlip for inverted microscope
Curity 4x4 inch all purpose sponge gauze Covidien 9024 Sponge
EGFPcyto mouse Jackson lab 003291 Mouse line
Fiji/ImageJ software Open source - Free software tool
Fine forceps Braintree Scientific, Inc FC003 8 Tissue forceps
Fluoview 1000 microscope Olympus FV1000 Confocal microscope
FluoView software Olympus - Confocal microscope and Two-photon microscope
Hand-held electric razor Braintree Scientific, Inc CLP-8786-451A Cordless clipper
Heat pad Braintree Scientific, Inc DPIP Heat pad for animals
HyD detectors Leica - Leica 4Tune spectral detector
Imaris software Bitplane / Oxford instruments - Commercial software tool
Ingisht X3 tunable laser Spectra Physics Insight X3 Tunable Pulse-Laser
Isoflurane VetOne 13985-046-40 Anesthetic
Ketamine  VetOne 13985-702-10 Anesthetic
LAS X Software Leica - Two-photon microscope software tool
Mai-Tai tunable laser Spectra Physics Mai-Tai Laser
MetaMorph Molecular Devices - Commercial software tool
Monodansylpentane AUTODOT Abcepta Sm1000a Lipid droplet dye
MPE-RS microscope Olympus IX70 Two-photon microscope
mT/mG mouse Jackson lab 007676 Mouse line
Objective heater Bioptechs 150819 Objective heater for both confocal and two-photon microscopes
Oxygen-saturated respiration chamber Patterson Scientific 78933385, SAS3 and EVAC4 Gas Anesthesia and evacuation system 
Parafilm Heathrow Scientific HS234526B Semi-transparent, flexible, thermoplastic film
PMT detector Olympus - Descanned   detectors
PMT detector LSM-Technology Custom built Non-Descanned Detectors
Pump Harvard Apparatus 703602, 704402 Nanomite injector and controller
Saline Quality Biological 114-055-721EA Normal saline
Sharp blunt-ended scissors Braintree Scientific, Inc SCT-S 508 Surgical scissors
Syringe Covidien 22-257-150 1mL tuberculin syringe
TCS SP8 Dive Spectral microscope Leica SP8 Two-photon microscope
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Ng, Y., Masedunskas, A., Heydecker, M., Ebrahim, S., Weigert, R., Mather, I. H. Intravital Subcellular Microscopy of the Mammary Gland. J. Vis. Exp. (184), e63674, doi:10.3791/63674 (2022).More

Ng, Y., Masedunskas, A., Heydecker, M., Ebrahim, S., Weigert, R., Mather, I. H. Intravital Subcellular Microscopy of the Mammary Gland. J. Vis. Exp. (184), e63674, doi:10.3791/63674 (2022).

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