Intravital Subcellular Microscopy of the Mammary Gland

Summary

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

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 cells^1,2, the progression of metastatic tumors^3,4,5, 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 lactation^8,9, and oxytocin-mediated contraction of myoepithelial cells^9,10. 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 ring^1,3,11. 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 tracing^1,12 and monitoring the invasion and progression of mammary tumors in situ^3,11. 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 spacers^4,9,13. 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 lumen^15,16, and the remodeling of the gland during involution^17,18.

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 EGFP_cyto FvB mouse was used, in which enhanced green fluorescent protein (EGFP) is targeted to the cytoplasm in most cells²⁰ (denoted EGFP_cyto), 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 tdTomato_membr (mT), and mice expressing EGFP, after excision of the tdTomato gene are denoted EGFP_membr (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.

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% isoflurane followed by decapitation. Pups ≥10 days of age should be euthanized via CO₂ inhalation 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 EGFP_cyto or EGFP_membr (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 tdTomato_membr (mT) or EGFP_membr (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 EGFP_cyto or EGFP_membr (mG) mouse labeled with BODIPY_665/676 (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 BODIPY_665/676 using 488 and 633 lasers, respectively; for EGFP, excitation 488 nm, and emission 560 nm, with band-pass filter BA 505-605; for BODIPY_665/676, 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 EGFP_membr (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 tdTomato_membr (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 via CO₂ inhalation following institutionally approved protocols.

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.

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 pr...

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 microscopes^29,30. In addition, the fluorophore is only excited at the focal point, which reduces light scatter...

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
Tweezers	Braintree Scientific, Inc	FC032	Tissue forceps
Xylazine	VetOne	13985-704-10	Anesthetic