Name: Intravital Subcellular Microscopy of the Mammary Gland (Video) | JoVE Journal
Uploaded: 2022-06-24T14:00:00.000+00:00
Duration: 4 min 14 s
Description: Learn intravital subcellular microscopy of the lactating mouse mammary gland using confocal and multiphoton imaging. This method enables real-time visualization of secretory epithelial cells and dynamic lipid droplet movement.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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.&#160;This research was supported [in part] by the Intramural Research Program of the NIH.

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&#39;s Guide for the Care and Use of Laboratory Animals, the US Public Health Service&#39;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
<ol>
	<li>Weigh the mouse on a top-loading balance to calculate the amount of anesthetic required.</li>
	<li>During and after applying anesthetics, maintain body temperature by placing the mouse on a warming pad.</li>
	<li>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.</li>
	<li>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 &#176;C, which drops to 34.5 &#176;C under anesthesia.</li>
	<li>Cross-foster the pups22 if donor mice at a similar stage of lactation are available; otherwise, euthanize the litter. 
	&#8203;NOTE: Pups &#60;10 days of age should be euthanized by exposure to 3.0% isoflurane followed by decapitation. Pups &#8805;10 days of age should be euthanized via CO2 inhalation followed by cervical dislocation.</li>
</ol>
2. Surgery procedure
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Remove superficial connective and adipose tissue by gently teasing the surface layer with fine forceps.</li>
	<li>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).</li>
	<li>If required, treat the gland with exogenous agents, e.g., pharmacological agents or organelle-specific dyes, by bathing the exposed gland during surgery. 
	&#8203;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 &#60;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).</li>
</ol>
3. Imaging preparation
<ol>
	<li>Carefully position the mouse with the abdominal side down on a heated (37 &#176;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.</li>
	<li>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).</li>
	<li>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).</li>
	<li>Insert a subcutaneous indwelling catheter under the dorsal skin attached to a tube, syringe, and pump (see Table of Materials).</li>
	<li>Confirm that the skin flap is stable with adequate blood flow by conventional fluorescence microscopy (Figure 1Bvi).</li>
	<li>Cover the mouse with sponge gauze (see Table of Materials) to keep warm during imaging, 
	&#8203;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.</li>
</ol>
4. Microscopy
<ol>
	<li>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 &#176;C (see Table of Materials).
	<ol>
		<li>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.</li>
		<li>Collect images by line scan at either 4 or 8 &#181;s/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).</li>
	</ol>
	</li>
	<li>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 &#176;C preheated 30x objective (see Table of Materials).
	<ol>
		<li>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.</li>
		<li>Collect images by line scan at 2 &#181;s/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.</li>
	</ol>
	</li>
	<li>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 &#176;C preheated 63x objective (see Table of Materials).
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
5. Euthanasia
<ol>
	<li>Euthanize the mice at the end of imaging via CO2 inhalation following institutionally approved protocols.</li>
</ol>
6. Creation of real-time videos
<ol>
	<li>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.</li>
</ol>

<ol><li>Scheele, C. L. G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identity+and+dynamics+of+mammary+stem+cells+during+branching+morphogenesis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Identity and dynamics of mammary stem cells during branching morphogenesis.</a> Nature. 542 (7641), 313-317 (2017).</li>
<li>Dawson, C. A., Mueller, S. N., Lindeman, G. J., Rios, A. C., Visvader, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+microscopy+of+dynamic+single-cell+behavior+in+mouse+mammary+tissue%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Intravital microscopy of dynamic single-cell behavior in mouse mammary tissue.</a> Nature Protocols. 16 (4), 1907-1935 (2021).</li>
<li>Kedrin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+imaging+of+metastatic+behavior+through+a+mammary+imaging+window%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Intravital imaging of metastatic behavior through a mammary imaging window.</a> Nature Methods. 5 (12), 1019-1021 (2008).</li>
<li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic%2C+long-term+in+vivo+imaging+of+tumor-stroma+interactions+in+mouse+models+of+breast+cancer+using+spinning-disk+confocal+microscopy%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">Dynamic, long-term in vivo imaging of tumor-stroma interactions in mouse models of breast cancer using spinning-disk confocal microscopy.</a> Cold Spring Harbor Protocols. (2), (2011).</li>
<li>Ellenbroek, S. I. J., van Rheenen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+hallmarks+of+cancer+in+living+mice%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">Imaging hallmarks of cancer in living mice.</a> Nature Reviews Cancer. 14 (6), 406-418 (2014).</li>
<li>Dawson, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-resident+ductal+macrophages+survey+the+mammary+epithelium+and+facilitate+tissue+remodelling%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">Tissue-resident ductal macrophages survey the mammary epithelium and facilitate tissue remodelling.</a> Nature Cell Biology. 22 (5), 546-558 (2020).</li>
<li>Ebrahim, S., Weigert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+microscopy+in+mammalian+multicellular+organisms%5BTitle%5D)+AND+%22Current+Opinion+in+Cell+Biology%22%5BJournal%5D)">Intravital microscopy in mammalian multicellular organisms.</a> Current Opinion in Cell Biology. 59, 97-103 (2019).</li>
<li>Masedunskas, A., Weigert, R., Mather, I. H. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMasedunskas%2C+A+%22Advances+in+Intravital+Microscopy%22">Advances in Intravital Microscopy</a>. Weigert, R. , Weigert) Springer-Verlag. Netherlands. 187-204 (2014).</li>
<li>Masedunskas, A., Chen, Y., Stussman, R., Weigert, R., Mather, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetics+of+milk+lipid+droplet+transport%2C+growth%2C+and+secretion+revealed+by+intravital+imaging%3A+lipid+droplet+release+is+intermittently+stimulated+by+oxytocin%5BTitle%5D)+AND+%22Molecular+Biology+of+the+Cell%22%5BJournal%5D)">Kinetics of milk lipid droplet transport, growth, and secretion revealed by intravital imaging: lipid droplet release is intermittently stimulated by oxytocin.</a> Molecular Biology of the Cell. 28 (7), 935-946 (2017).</li>
<li>Stevenson, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiscale+imaging+of+basal+cell+dynamics+in+the+functionally-mature+mammary+gland%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Multiscale imaging of basal cell dynamics in the functionally-mature mammary gland.</a> Proceedings of the National Academy of Sciences of the United States of America. 117 (43), 26822-26832 (2020).</li>
<li>Shan, S., Sorg, B., Dewhirst, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+rodent+mammary+window+of+orthotopic+breast+cancer+for+intravital+microscopy%5BTitle%5D)+AND+%22Microvascular+Research%22%5BJournal%5D)">A novel rodent mammary window of orthotopic breast cancer for intravital microscopy.</a> Microvascular Research. 65 (2), 109-117 (2003).</li>
<li>Zomer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brief+report%3A+Intravital+imaging+of+cancer+stem+cell+plasticity+in+mammary+tumors%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Brief report: Intravital imaging of cancer stem cell plasticity in mammary tumors.</a> Stem Cells. 31 (3), 602-606 (2013).</li>
<li>Harper, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+of+early+dissemination+and+metastasis+in+Her2%2B+mammary+cancer%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Mechanism of early dissemination and metastasis in Her2+ mammary cancer.</a> Nature. 540 (7634), 588-592 (2016).</li>
<li>Burgoyne, R. D., Duncan, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Secretion+of+milk+proteins%5BTitle%5D)+AND+%22Journal+of+Mammary+Gland+Biology+and+Neoplasia%22%5BJournal%5D)">Secretion of milk proteins.</a> Journal of Mammary Gland Biology and Neoplasia. 3 (3), 275-286 (1998).</li>
<li>Monks, J., Neville, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Albumin+transcytosis+across+the+epithelium+of+the+lactating+mouse+mammary+gland%5BTitle%5D)+AND+%22Journal+of+Physiology+London%22%5BJournal%5D)">Albumin transcytosis across the epithelium of the lactating mouse mammary gland.</a> Journal of Physiology London. 560, 267-280 (2004).</li>
<li>Boisgard, R., Chanat, E., Lavialle, F., Pauloin, A., Ollivier-Bousquet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Roads+taken+by+milk+proteins+in+mammary+epithelial+cells%5BTitle%5D)+AND+%22Livestock+Production+Science%22%5BJournal%5D)">Roads taken by milk proteins in mammary epithelial cells.</a> Livestock Production Science. 70 (1-2), 49-61 (2001).</li>
<li>Green, K. A., Lund, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ECM+degrading+proteases+and+tissue+remodelling+in+the+mammary+gland%5BTitle%5D)+AND+%22Bioessays%22%5BJournal%5D)">ECM degrading proteases and tissue remodelling in the mammary gland.</a> Bioessays. 27 (9), 894-903 (2005).</li>
<li>Lund, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+distinct+phases+of+apoptosis+in+mammary+gland+involution%3A+proteinase-independent+and+-dependent+pathways%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways.</a> Development. 122 (1), 181-193 (1996).</li>
<li>Abe, T., Fujimori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reporter+mouse+lines+for+fluorescence+imaging%5BTitle%5D)+AND+%22Develoment+Growth+and+Differentiation%22%5BJournal%5D)">Reporter mouse lines for fluorescence imaging.</a> Develoment Growth and Differentiation. 55 (4), 390-405 (2013).</li>
<li>Hadjantonakis, A. K., Gertsenstein, M., Ikawa, M., Okabe, M., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generating+green+fluorescent+mice+by+germline+transmission+of+green+fluorescent+ES+cells%5BTitle%5D)+AND+%22Mechanisms+of+Development%22%5BJournal%5D)">Generating green fluorescent mice by germline transmission of green fluorescent ES cells.</a> Mechanisms of Development. 76 (1-2), 79-90 (1998).</li>
<li>Mazumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+global+double-fluorescent+Cre+reporter+mouse%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45 (9), 593-605 (2007).</li>
<li>Fostering litters. The Jackson Laboratory. , Available from: 
 <a target="_blank" href="https://www.jax.org/jax-mice-and-services/customer-support/technical-support/breeding-and-husbandry-support/general-husbandry-tips#">https://www.jax.org/jax-mice-and-services/customer-support/technical-support/breeding-and-husbandry-support/general-husbandry-tips#</a> (2022).</li>
<li>Yang, H. -J., Hsu, C. -L., Yang, J. -Y., Yang, W. -Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monodansylpentane+as+a+blue-fluorescent+lipid-droplet+marker+for+multi-color+live-cell+imaging%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Monodansylpentane as a blue-fluorescent lipid-droplet marker for multi-color live-cell imaging.</a> PloS One. 7 (3), 32693(2012).</li>
<li>Ebrahim, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+polyhedral+actomyosin+lattices+remodel+micron-scale+curved+membranes+during+exocytosis+in+live+mice%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">Dynamic polyhedral actomyosin lattices remodel micron-scale curved membranes during exocytosis in live mice.</a> Nature Cell Biology. 21 (8), 933-939 (2019).</li>
<li>Meyer, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+predictive+3D+multi-scale+model+of+biliary+fluid+dynamics+in+the+liver+lobule%5BTitle%5D)+AND+%22Cell+Systems%22%5BJournal%5D)">A predictive 3D multi-scale model of biliary fluid dynamics in the liver lobule.</a> Cell Systems. 4 (3), 277-290 (2017).</li>
<li>Macias, H., Hinck, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mammary+gland+development%5BTitle%5D)+AND+%22Wiley+Interdisciplinary+Reviews+Developmental+Biology%22%5BJournal%5D)">Mammary gland development.</a> Wiley Interdisciplinary Reviews Developmental Biology. 1 (4), 533-557 (2012).</li>
<li>Mather, I. H., Masedunskas, A., Chen, Y., Weigert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Symposium+review%3A+Intravital+imaging+of+the+lactating+mammary+gland+in+live+mice+reveals+novel+aspects+of+milk-lipid+secretion%5BTitle%5D)+AND+%22Journal+of+Dairy+Science%22%5BJournal%5D)">Symposium review: Intravital imaging of the lactating mammary gland in live mice reveals novel aspects of milk-lipid secretion.</a> Journal of Dairy Science. 102 (3), 2760-2782 (2019).</li>
<li>Caspi, A., Granek, R., Elbaum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+diffusion+in+active+intracellular+transport%5BTitle%5D)+AND+%22Physical+Review+Letters%22%5BJournal%5D)">Enhanced diffusion in active intracellular transport.</a> Physical Review Letters. 85, 5655-5658 (2000).</li>
<li>Zipfel, W. R., Williams, R. M., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonlinear+magic%3A+multiphoton+microscopy+in+the+biosciences%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Nonlinear magic: multiphoton microscopy in the biosciences.</a> Nature Biotechnology. 21 (11), 1369-1377 (2003).</li>
<li>Weigert, R., Porat-Shliom, N., Amornphimoltham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+cell+biology+in+live+animals%3A+Ready+for+prime+time%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">Imaging cell biology in live animals: Ready for prime time.</a> Journal of Cell Biology. 201 (7), 969-979 (2013).</li>
<li>So, P. T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-photon+fluorescence+light+microscopy%5BTitle%5D)+AND+%22Encyclopedia+of+Life+Sciences%22%5BJournal%5D)">Two-photon fluorescence light microscopy.</a> Encyclopedia of Life Sciences. , 1-5 (2002).</li>
<li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+of+vital+signs+for+long-term+survival+of+mice+under+anesthesia%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">Monitoring of vital signs for long-term survival of mice under anesthesia.</a> Cold Spring Harbor Protocols. 2011 (2), 5563(2011).</li>
<li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+of+mice+for+long-term+intravital+imaging+of+the+mammary+gland%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">Preparation of mice for long-term intravital imaging of the mammary gland.</a> Cold Spring Harbor Protocols. 2011 (2), 5562(2011).</li>
<li>Nishinakagawa, H., Mochizuki, K., Nishida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+blood+supply+to+the+mammary+glands+of+the+mouse%2C+rat%2C+hamster+and+guinea-pig%5BTitle%5D)+AND+%22Japanese+Journal+of+Zoological+Science%22%5BJournal%5D)">On the blood supply to the mammary glands of the mouse, rat, hamster and guinea-pig.</a> Japanese Journal of Zoological Science. 39 (7), 283-291 (1968).</li>
<li>Parslow, A., Cardona, A., Bryson-Richardson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sample+drift+correction+following+4D+confocal+time-lapse+imaging%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Sample drift correction following 4D confocal time-lapse imaging.</a> Journal of Visualized Experiments. (86), e51086(2014).</li>
<li>Palade, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+aspects+of+the+process+of+protein+synthesis%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Intracellular aspects of the process of protein synthesis.</a> Science. 189 (4200), 347-358 (1975).</li>
<li>Heald, C. W., Saacke, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytological+comparison+of+milk+protein+synthesis+of+rat+mammary+tissue+in+vivo+and+in+vitro%5BTitle%5D)+AND+%22Journal+of+Dairy+Science%22%5BJournal%5D)">Cytological comparison of milk protein synthesis of rat mammary tissue in vivo and in vitro.</a> Journal of Dairy Science. 55 (5), 621-628 (1972).</li>
<li>Hunziker, W., Kraehenbuhl, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epithelial+transcytosis+of+immunoglobulins%5BTitle%5D)+AND+%22Journal+of+Mammary+Gland+Biology+and+Neoplasia%22%5BJournal%5D)">Epithelial transcytosis of immunoglobulins.</a> Journal of Mammary Gland Biology and Neoplasia. 3 (3), 287-302 (1998).</li>
<li>Messal, H. A., van Rheenen, J., Scheele, C. L. G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+intravital+microscopy+toolbox+to+study+mammary+gland+dynamics+from+cellular+level+to+organ+scale%5BTitle%5D)+AND+%22Journal+of+Mammary+Gland+Biology+and+Neoplasia%22%5BJournal%5D)">An intravital microscopy toolbox to study mammary gland dynamics from cellular level to organ scale.</a> Journal of Mammary Gland Biology and Neoplasia. 26 (1), 9-27 (2021).</li>
<li>Teter, B. B., Sampugna, J., Keeney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Milk+fat+depression+in+C57Bl%2F6J+mice+consuming+partially+hydrogenated+fat%5BTitle%5D)+AND+%22Journal+of+Nutrition%22%5BJournal%5D)">Milk fat depression in C57Bl/6J mice consuming partially hydrogenated fat.</a> Journal of Nutrition. 120 (8), 818-824 (1990).</li>
<li>Russell, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transduction+of+the+mammary+epithelium+with+adenovirus+vectors+in+vivo%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">Transduction of the mammary epithelium with adenovirus vectors in vivo.</a> Journal of Virology. 77 (10), 5801-5809 (2003).</li>
</ol>

None of the authors have any conflicting interests to declare.

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 scatter...

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&#39;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 &#60;1 &#181;m, 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.

<table><tbody><tr><td>488 laser</td><td>Melles-Griot</td><td>-</td><td>CW&amp;nbsp; laser 50 mW</td></tr><tr><td>60x PLAPON oil immersion objective (NA 1.42)</td><td>Olympus</td><td>1-U2B933</td><td>Lens Confocal microscope</td></tr><tr><td>633 laser</td><td>Melles-Griot</td><td>-</td><td>CW He-Ne laser 12 mW</td></tr><tr><td>63x objective (NA 1.40, HC PL APO CS2)</td><td>Leica</td><td>11506350</td><td>Lens Two-photon microscope</td></tr><tr><td>BA 410-460 nm</td><td>Chroma</td><td>-</td><td>Band-pass filter</td></tr><tr><td>BA 495-540 nm</td><td>Chroma</td><td>-</td><td>Band-pass filter</td></tr><tr><td>BA 505-605 nm</td><td>Chroma</td><td>-</td><td>Band-pass filter</td></tr><tr><td>BA 655-755 nm</td><td>Chroma</td><td>-</td><td>Band-pass filter</td></tr><tr><td>Boron-dipyrromethane (BODIPY) 665/676</td><td>Thermo Fisher Scientific</td><td>B3932</td><td>Lipid peroxiation sensor</td></tr><tr><td>Carbomer-940</td><td>Snowdrift Farm</td><td>739601480651</td><td>Gel</td></tr><tr><td>Catheter</td><td>Terumo</td><td>SV27EL</td><td>Winged infusion sets&amp;nbsp;</td></tr><tr><td>Cauterizer&amp;nbsp;</td><td>Braintree Scientific, Inc</td><td>GEM 5917</td><td>Cautery system</td></tr><tr><td>CMV-Cre mouse&amp;nbsp;</td><td>Jackson lab</td><td>006054</td><td>Mouse line</td></tr><tr><td>Coverslip</td><td>Bioptechs</td><td>-</td><td>30mm diameter coverlip for inverted microscope</td></tr><tr><td>Curity 4x4 inch all purpose sponge gauze</td><td>Covidien</td><td>9024</td><td>Sponge</td></tr><tr><td>EGFP&lt;sub&gt;cyto&lt;/sub&gt; mouse</td><td>Jackson lab</td><td>003291</td><td>Mouse line</td></tr><tr><td>Fiji/ImageJ software</td><td>Open source</td><td>-</td><td>Free software tool</td></tr><tr><td>Fine forceps</td><td>Braintree Scientific, Inc</td><td>FC003 8</td><td>Tissue forceps</td></tr><tr><td>Fluoview 1000 microscope</td><td>Olympus</td><td>FV1000</td><td>Confocal microscope</td></tr><tr><td>FluoView software</td><td>Olympus</td><td>-</td><td>Confocal microscope and Two-photon microscope</td></tr><tr><td>Hand-held electric razor</td><td>Braintree Scientific, Inc</td><td>CLP-8786-451A</td><td>Cordless clipper</td></tr><tr><td>Heat pad</td><td>Braintree Scientific, Inc</td><td>DPIP</td><td>Heat pad for animals</td></tr><tr><td>HyD detectors</td><td>Leica</td><td>-</td><td>Leica 4Tune spectral detector</td></tr><tr><td>Imaris software</td><td>Bitplane / Oxford instruments</td><td>-</td><td>Commercial software tool</td></tr><tr><td>Ingisht X3 tunable laser</td><td>Spectra Physics</td><td>Insight X3</td><td>Tunable Pulse-Laser</td></tr><tr><td>Isoflurane</td><td>VetOne</td><td>13985-046-40</td><td>Anesthetic</td></tr><tr><td>Ketamine&amp;nbsp;</td><td>VetOne</td><td>13985-702-10</td><td>Anesthetic</td></tr><tr><td>LAS X Software</td><td>Leica</td><td>-</td><td>Two-photon microscope software tool</td></tr><tr><td>Mai-Tai tunable laser</td><td>Spectra Physics</td><td>Mai-Tai</td><td>Laser</td></tr><tr><td>MetaMorph</td><td>Molecular Devices</td><td>-</td><td>Commercial software tool</td></tr><tr><td>Monodansylpentane AUTODOT</td><td>Abcepta</td><td>Sm1000a</td><td>Lipid droplet dye</td></tr><tr><td>MPE-RS microscope</td><td>Olympus</td><td>IX70</td><td>Two-photon microscope</td></tr><tr><td>mT/mG mouse</td><td>Jackson lab</td><td>007676</td><td>Mouse line</td></tr><tr><td>Objective heater</td><td>Bioptechs</td><td>150819</td><td>Objective heater for both confocal and two-photon microscopes</td></tr><tr><td>Oxygen-saturated respiration chamber</td><td>Patterson Scientific</td><td>78933385, SAS3 and EVAC4</td><td>Gas Anesthesia and evacuation system&amp;nbsp;</td></tr><tr><td>Parafilm</td><td>Heathrow Scientific</td><td>HS234526B</td><td>Semi-transparent, flexible, thermoplastic film</td></tr><tr><td>PMT detector</td><td>Olympus</td><td>-</td><td>Descanned&amp;nbsp;&amp;nbsp; detectors</td></tr><tr><td>PMT detector</td><td>LSM-Technology</td><td>Custom built</td><td>Non-Descanned Detectors</td></tr><tr><td>Pump</td><td>Harvard Apparatus</td><td>703602, 704402</td><td>Nanomite injector and controller</td></tr><tr><td>Saline</td><td>Quality Biological</td><td>114-055-721EA</td><td>Normal saline</td></tr><tr><td>Sharp blunt-ended scissors</td><td>Braintree Scientific, Inc</td><td>SCT-S 508</td><td>Surgical scissors</td></tr><tr><td>Syringe</td><td>Covidien</td><td>22-257-150</td><td>1mL tuberculin syringe</td></tr><tr><td>TCS SP8 Dive Spectral microscope</td><td>Leica</td><td>SP8</td><td>Two-photon microscope</td></tr><tr><td>Tweezers&amp;nbsp;</td><td>Braintree Scientific, Inc</td><td>FC032</td><td>Tissue forceps</td></tr><tr><td>Xylazine&amp;nbsp;</td><td>VetOne</td><td>13985-704-10</td><td>Anesthetic</td></tr></tbody></table>

intravital subcellular microscopy of the mammary gland

Intravital Subcellular Microscopy of the Mammary Gland

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&#160;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 &#8805; 0.7 &#181;m. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 &#181;m 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.

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 ...

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

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...

intravital-subcellular-microscopy-of-the-mammary-gland

Research

JoVE Journal

Biology

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

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.

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Intraductal Delivery to the Rabbit Mammary Gland

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin. These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

Longitudinal Intravital Imaging Through Clear Silicone Windows

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.

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The present protocol describes intraductal injection of viral vectors via the teat to deliver genes of interest into the mammary epithelial cells.

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

Learn intravital subcellular microscopy of the lactating mouse mammary gland using confocal and multiphoton imaging. This method enables real-time visualization of secretory epithelial cells and dynamic lipid droplet movement.

Intravital Subcellular Microscopy of the Mammary Gland

Explore More Videos