The authors would like to acknowledge April Wiles and Dr. Roger Broderson for assistance with animal necropsy and gland isolation, respectively. Funding for this work was supported by the Philadelphia College of Osteopathic Medicine Centers for Chronic Disorders of Aging.

Procedures involving animal subjects in this protocol were reviewed and approved by the Institutional Animal Care and Use Committee of the Philadelphia College of Osteopathic Medicine and all techniques were conducted under strict ethical guidelines.
1. Sample Procurement and Processing
<ol>
	<li>Select appropriate animal subject and place into a CO2 chamber. For this experiment, use 8-10 week old female nulliparous B6.Cg-Cav1tmMIs/J and C57BI/6J.</li>
	<li>Turn on gas flow to 30-40%. Once the animal is visibly unconscious after about 2 min, open the gas valve to full pressure for an additional 5 min. 
	NOTE: Confirm animal death by observing visible signs of breathing (e.g. movement of the chest) for a period of 10 min after cessation of CO2 delivery. If the animal is still alive, it may be placed back in the CO2 chamber. Cervical dislocation may also be used although it is not recommended as blood may accumulate around the mammary glands interfering with the dissection and results.</li>
	<li>Following sacrifice, pin carcass in a supine position and saturate with 70% ethanol.</li>
	<li>Pinch the pelt just above the pubis using forceps and nick with small surgical scissors. Rotating the scissors, cut the pelt along the ventral midline moving caudal to cranial.</li>
	<li>With larger scissors or a hemostat, bluntly dissect the subcutaneous fascia bilaterally, using caution not to puncture the peritoneum. Cut along the horizontal margins at both distal ends of the incision.</li>
	<li>Pin the pelt flaps open and spray again with 70% ethanol. 
	NOTE: Although the use of 70% ethanol permitted better visual distinction between the gland and the surrounding subcutaneous tissue, be careful to avoid drying of the tissue as a result of use of this solution. To avoid drying, remove the gland in a time frame not to exceed 4 min. As an alternative to 70% ethanol, the investigator may also substitute a general isolate buffer, such as 1x PBS, to avoid tissue desiccation.</li>
	<li>Locating the mammary glands of interest, slide a #4 scalpel blade along the inside of the pelt flaps, cutting the mammary gland and associated cutaneous adipose free from the dermis. 
	NOTE: 3.5x surgical micro loupes may aid in easier visualization of the glands.</li>
	<li>Immediately submerge newly isolated gland in conical tube containing 10:1 solution-tissue volume of 10% neutral buffered formalin for 24-48 h. 
	NOTE: Depending on intent of study, many alternative fixatives may be used such as paraformaldehyde, ethanol for genomic studies, and commercially available RNA preserving buffers.</li>
	<li>Follow institutional protocols for paraffin embedding, submit samples to a vendor for processing, embedding, and slicing/slide mounting. Section tissues at 5 &#181;m. 
	NOTE: Due to excess adipose surrounding gland, consider removing 100-200 &#181;m of tissue before sectioning and staining.</li>
</ol>
2. Tissue Staining
<ol>
	<li>Immunohistochemistry
	<ol>
		<li>Place slides to be stained on heat block set at 58 &#730;C for 1 h to melt paraffin. 
		CAUTION: Melting paraffin wax may run off slide. Monitor closely or place wipe under slide to capture runoff wax. 
		NOTE: This step is not essential and may be skipped. If results are not as expected, adding this step back may improve results.</li>
		<li>Incubate slides in a slide jar containing 100% xylene for 30 min ensuring complete submersion. Repeat once. 
		NOTE: At this point, visual inspection should be performed to ensure that tissue sections are free of adipose tissue and paraffin. If additional adipose tissue or wax is evident, the slide should be re-submerged in xylene. Use caution to minimize added exposure to xylene as this can cause shrinkage of the tissue.</li>
		<li>Rehydrate tissues in a slide jar containing 100% ethanol for 10 min. Repeat once.</li>
		<li>Move slides to a slide jar containing 95% ethanol for 10 min. Repeat once.</li>
		<li>Move slides to a slide jar containing 75% ethanol for 5 min.</li>
		<li>Move slides to a slide jar containing 50% ethanol for 5 min.</li>
		<li>Boil 10 mM sodium citrate solution (pH 6.0) in a hot plate or a microwave and pour into Coplin jar. 
		CAUTION: Carefully monitor solution while heating and take care to avoid boil over. Container will be very hot. Use protection to avoid burns.</li>
		<li>Slowly place slides into Coplin jar, ensuring complete submersion, and incubate at 100 &#176;C for 10 min to retrieve epitopes. Remove and carefully dry slides.</li>
		<li>Draw a barrier around tissue with a hydrophobic marker.Add enough endogenous enzyme blocker (hydrogen peroxide and sodium azide, available commercially) to cover tissue and incubate for 10 min.</li>
		<li>Submerge slides in 1x phosphate buffered saline (PBS) for 10 min.</li>
		<li>Add enough 10% donkey serum in 1x PBS to cover tissue and incubate for 1 h at room temperature in a humidified chamber. 
		NOTE: The experiment can be paused here by storing the slides in the humidified chamber at 4 &#176;C overnight. While donkey serum was found to yield optimal staining results, the investigator may wish to test different sera such as bovine serum albumin (BSA), goat serum or horse serum to determine optimal results. If using BSA, the investigator should prepare this fresh before use.</li>
		<li>Decant excess blocker from slides and add properly diluted primary antibody in 1% serum directly to slides ensuring that the tissue is evenly covered in solution. Incubate for 30 min at room temperature in a humidified chamber. 
		NOTE: This step may be continued for longer time durations with humid chamber stored at 4 &#176;C. Make sure to include proper negative control slides (e.g. a slide with no primary antibody, known antigen-negative tissue, etc.) at this step.</li>
		<li>Carefully wash slides by flowing about 1 mL of diH2O over tissue and incubate in 1 change of 1x PBS for 5 min.</li>
		<li>Decant excess PBS and add enough horseradish peroxidase label to cover tissue and incubate for 30 min in a humidified chamber. 
		NOTE: At this step, the investigator may wish to proceed to fluorescent staining using fluorescently conjugated secondary antibodies. If this is desired, the next steps described should be modified accordingly.</li>
		<li>Carefully wash slides by flowing about 1 mL of diH2O over tissue and incubate in 1 change of 1x PBS for 5 min.</li>
		<li>Make diaminobenzidine (DAB) plus chromogen solution according to the manufacturer suggested ratio and mix by vortex. 
		NOTE: Many ready-made kits are commercially available aside from the one used in this protocol.</li>
		<li>Decant excess buffer from slides and add 2-3 drops (10-50 &#181;L) of chromogen stain directly to tissues and incubate for 5 min in humid chamber. Carefully wash slides by flowing about 1 mL diH2O over tissue. 
		CAUTION: DAB-chromogen is highly toxic. Avoid direct contact of stain with skin and collect flow off in hazardous waste.</li>
	</ol>
	</li>
	<li>Hematoxylin and Eosin (H&#38;E)
	<ol>
		<li>Following final rinse after chromogen incubation, submerge slides in Harris hematoxylin for 2-2.5 min. Rinse slides gently under tap water for 1-2 min. 
		NOTE: Take care to avoid direct jet of water onto tissues.</li>
		<li>Submerge slides for 2-3 s in differentiation solution (0.25 mL of hydrochloric acid in 100 mL of 70% ethanol). Rinse slides gently under tap water for about 1-2 min.</li>
		<li>Submerge slides in blue agent (4.5 mg calcium carbonate in 100 mL tap water, pH adjusted to 9.4) for 60 s. Rinse slides in 95% ethanol for 30 s.</li>
		<li>Submerge slides in alcoholic eosin Y for 2-3 min.</li>
		<li>Dehydrate tissue in 2 changes of 95% ethanol for 1 min each.</li>
		<li>Dehydrate tissue in 1 change of 100% ethanol for 1 min.</li>
		<li>Carefully dry slides with a lint-free wipe. Apply 1 drop (about 100-200 &#181;L) of synthetic, non-aqueous, resin-based mounting media to slide and apply coverslip. 
		NOTE: If a different staining method was elected, a different mounting media may be required. For instance, if the investigator chose a fluorescent-conjugated secondary antibody, an aqueous mount would be more ideal.</li>
		<li>Allow slides to set overnight at room temperature.</li>
	</ol>
	</li>
	<li>Picrosirius Red (PSR) Staining
	<ol>
		<li>Select slides to be stained and follow immunohistochemistry protocol through step 6 (50% ethanol soak).</li>
		<li>Submerge slides in PSR stain for 1 h.</li>
		<li>Submerge slides in 0.5% acetic acid for 1-2 s, twice to differentiate stain.</li>
		<li>Dehydrate tissues in 2 changes of 95% ethanol for 1 min each.</li>
		<li>Dehydrate tissues in 1 change of 100% ethanol for 1 min.</li>
		<li>Clear slides by briefly submerging in 100% xylene for about 3-4 s.</li>
		<li>Carefully dry slides with a lint-free wipe. Apply 1 drop (about 100-200 &#181;L) of synthetic, non-aqueous, resin-based mounting media to slide and apply coverslip.</li>
	</ol>
	</li>
</ol>
3. Sample Analysis
<ol>
	<li>Ductal Analysis
	<ol>
		<li>Select slides stained for &#945;-SMA. Using a light microscope fitted with a camera-mounted objective, collect representative images at 20X magnification.</li>
		<li>Using ImageJ (NIH), distinguish and count ducts in each image. These can be enumerated manually or one may assign a numerical score to individual ducts. To assign a numerical score, open ImageJ and select Plugins. Next select Analyze and choose Cell Counter from the drop down menu. Click Initialize then highlight Type 1 to begin labeling ducts. When finished, select Results to view the ductal count. 
		NOTE: Take care to verify structures are ductal and not vascular.</li>
		<li>In &#39;Set Measurement&#39; options, check &#39;Perimeter&#39; and &#39;Area&#34;.</li>
		<li>Using the freehand polygon tool, draw a line around each duct at the myoepithelial compartment (visibility aided by &#945;-SMA stain). Select &#39;Measure&#39; and record the perimeter and area.</li>
		<li>Again using the freehand polygon tool, draw a line along the apical side of the ductal epithelium within the interior of the lumen. Select &#39;Measure&#39; and record the perimeter and area.</li>
		<li>Subtract the perimeter of the luminal compartment from the perimeter of the myoepithelial compartment in order to obtain the circumference of the ductal epithelium. Subtract the area of the luminal compartment from the area of the myoepithelial compartment to obtain the area of the ductal epithelium.</li>
	</ol>
	</li>
	<li>Immunohistochemistry Analysis
	<ol>
		<li>Upload brightfield images to an analytical software, such as ImageJ or FIJI Suite. 
		NOTE: This protocol outlines the steps for staining analysis using ImageJ and the IHC Toolbox plugin.</li>
		<li>Open the IHC Toolbox from the Plugin menu. In the &#34;Select Model&#34; combo box that opens, select appropriate stain (i.e. H-DAB, PSR, etc.). Select the &#34;Color&#34; option to isolate the stain. This will open a result window. 
		NOTE: This method is appropriate if a protein of interest is located in the extracellular or cytosolic spaces, or is bound to the plasma membrane. If the protein of interest is nuclear, selecting &#34;Nuclei&#34; will prompt the plugin to analyze the image for positively stained nuclei.</li>
		<li>Use the Color Chooser Slide to ensure proper isolation of the stain without background inclusion or excessive stain exclusion. 
		NOTE: If the automatic mode is unable to detect the stain or does not result in acceptable images, draw a square Region of Interest (ROI) over an identified stained region. On the IHC Toolbox window, select &#34;Train&#34; to direct the plugin to the appropriate target.</li>
		<li>Convert the image to 16-bit. Go to Image &#8594; Type &#8594;&#160;16 bit.</li>
		<li>Threshold the image. Go to Image&#160;&#8594;&#160;Adjust &#8594;&#160;Auto Threshold.</li>
		<li>Set the image measurement scale by drawing a line ROI directly over the scale bar in the image then assigning the length. To set the scale bar, go to Analyze &#8594;&#160;Set Scale. Input the number of pixels per the desired unit of length (e.g. 120 pixels per 50 &#181;m).</li>
		<li>Measure the resultant area and mean grey value. Go to Analyze &#8594;&#160;Set Measurements and collect the measurement by clicking Analyze &#8594;&#160;Measure.</li>
	</ol>
	</li>
</ol>

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+reorganization+at+the+tumor-stromal+interface+facilitates+local+invasion.">Collagen reorganization at the tumor-stromal interface facilitates local invasion.</a> BMC Med. 4 (1), 38 (2006).</li><li>Provenzano, P. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+density+promotes+mammary+tumor+initiation+and+progression.">Collagen density promotes mammary tumor initiation and progression.</a> BMC Med. 6, 11 (2008).</li><li>Cox, T. R., Erler, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+and+homeostasis+of+the+extracellular+matrix:+implications+for+fibrotic+diseases+and+cancer.">Remodeling and homeostasis of the extracellular matrix: implications for fibrotic diseases and cancer.</a> Dis Model Mech. 4 (2), 165-178 (2011).</li><li>Ioachim, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+expression+of+extracellular+matrix+components+tenascin,+fibronectin,+collagen+type+IV+and+laminin+in+breast+cancer:+their+prognostic+value+and+role+in+tumour+invasion+and+progression.">Immunohistochemical expression of extracellular matrix components tenascin, fibronectin, collagen type IV and laminin in breast cancer: their prognostic value and role in tumour invasion and progression.</a> Eur J Cancer. 38 (18), 2362-2370 (2002).</li><li>Barkan, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+growth+from+dormant+cells+induced+by+a+col-I-enriched+fibrotic+environment.">Metastatic growth from dormant cells induced by a col-I-enriched fibrotic environment.</a> Cancer Res. 70 (14), 5706-5716 (2010).</li><li>Levental, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139 (5), 891-906 (2009).</li><li>Wang, J. 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L., Speirs, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+functional+significance+of+loss+of+caveolin-1+expression+in+breast+cancer-associated+fibroblasts.">Clinical and functional significance of loss of caveolin-1 expression in breast cancer-associated fibroblasts.</a> J Pathol. 227 (4), 490-498 (2012).</li><li>Witkiewicz, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+absence+of+stromal+caveolin-1+expression+predicts+early+tumor+recurrence+and+poor+clinical+outcome+in+human+breast+cancers.">An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor clinical outcome in human breast cancers.</a> Am J Pathol. 174 (6), 2023-2034 (2009).</li><li>Macias, H., Hinck, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+gland+development.">Mammary gland development.</a> Wiley Interdiscip Rev Dev Biol. 1 (4), 533-557 (2012).</li><li>Caligioni, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+reproductive+status/stages+in+mice.">Assessing reproductive status/stages in mice.</a> Curr Protoc Neurosci. , (2009).</li><li>Cannata, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+circulating+IGF-I+promotes+mammary+gland+development+and+proliferation.">Elevated circulating IGF-I promotes mammary gland development and proliferation.</a> Endocrinology. 151 (12), 5751-5761 (2010).</li><li>Thompson, C., Rahim, S., Arnold, J., Hielscher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+caveolin-1+alters+extracellular+matrix+protein+expression+and+ductal+architecture+in+murine+mammary+glands.">Loss of caveolin-1 alters extracellular matrix protein expression and ductal architecture in murine mammary glands.</a> PLoS One. 12 (2), 0172067 (2017).</li><li>Williams, C. 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The authors have nothing to disclose.

In the paper, we have described a technique to isolate intact mouse mammary glands for downstream histological analyses of ECM expression and ductal morphology. With respect to analyses of ductal morphology, this methodology enables the rapid investigation of ductal architecture based off of stained histological sections. Other methods of ductal analyses rely on injections of dyes to enable visualization of the ductal tree, methods which may be technically challenging and time consuming.
In br...

Breast cancer is characterized by a substantial degree of tissue fibrosis1,2,3,4. Referred to as the ECM, this non-cellular entity is found in varying degrees in all tissues and is primarily comprised of a complex meshwork of fibrillar and non-fibrillar collagens, elastin, and glycoproteins in addition to various signaling molecules that are sequestered in this matrix. Under homeostatic conditions, the deposition and degradation of the ECM is tightly controlled.5 During breast tumorigenesis, the balance of ECM deposition and degradation is disrupted. As such, breast tumors have been reported to express abundant ECM proteins such as collagens, fibronectin and tenascin-C amongst others.6 The abnormal expression of these proteins in addition to increased patterns of matrix crosslinking has been documented to promote breast tumor progression, metastasis and therapy resistance1,3,4,7,8,9.
To assess ECM composition and ductal morphology, isolation of intact mammary glands was performed. Here, we used female nulliparous mice deficient for caveolin-1, an integral membrane protein which has been linked to an aggressive breast tumor signature10,11,12, and control female nulliparous B6 mice. Histological processing and staining of these tissues permitted the identification of several ECM proteins along with characterization of ductal morphology.
Overall, the isolation of whole, intact mammary glands gives researchers the opportunity to investigate tissue-wide morphological or cellular changes occurring in response to exogenous or endogenous factors. Drawbacks of the technique are associated with analyses of 2 dimension (2D) tissue sections as opposed to a 3D perspective, which would yield a more complete picture of the complex morphology of the ductal tree. Given the complexity of cell-cell and cell-ECM interactions that take place in the mammary gland, the isolation of whole, intact glands is advantageous for efficiently analyzing ductal morphology and protein expression in various regions of the murine mammary gland.

<table>
	<tbody>
		<tr>
			<td>Light Microscope</td>
			<td>Olympus</td>
			<td>BX43</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Camera</td>
			<td>Olympus</td>
			<td>DP73</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td>Olympus</td>
			<td>cellSens Entry software</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>NIH</td>
			<td>ImageJ</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5x-R Surgical Micro Loupes</td>
			<td>Rose Micro Solutions</td>
			<td></td>
			<td>Magnification at researcher&#39;s preference</td>
		</tr>
		<tr>
			<td>Mayo Scissors</td>
			<td>Medline</td>
			<td>DYND04035</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining Rack</td>
			<td>Fisher Scientific</td>
			<td>121</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining Dish</td>
			<td>Fisher Scientific</td>
			<td>112</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin Jars</td>
			<td>Fisher Scientific</td>
			<td>19-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td>Size appropriate for tissue</td>
		</tr>
		<tr>
			<td>IHC EnVision+ Kit (HRP, Mouse, DAB+)</td>
			<td>Dako</td>
			<td>K400611-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrosirius Red Kit</td>
			<td>Abcam</td>
			<td>AB150681</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y, alcoholic</td>
			<td>Sigma-Aldrich</td>
			<td>HT110132</td>
			<td></td>
		</tr>
		<tr>
			<td>Harris Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>HHS16</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Serum</td>
			<td>EMD Millipore</td>
			<td>S30</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes, Reagent Grade</td>
			<td>Sigma-Aldrich</td>
			<td>214736</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 200 proof</td>
			<td>Sigma-Aldrich</td>
			<td>792780</td>
			<td>suitable for molecular biology</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline, 1x</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Fisher Scientific</td>
			<td>S279-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>202932</td>
			<td></td>
		</tr>
		<tr>
			<td>Permanent Mounting Medium</td>
			<td>Dako</td>
			<td>S1964</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt&#39;s Mounting Medium</td>
			<td>Sigma-Aldrich</td>
			<td>3989</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin antibody</td>
			<td>Abcam</td>
			<td>AB23750</td>
			<td></td>
		</tr>
		<tr>
			<td>Tenascin-C antibody</td>
			<td>Abcam</td>
			<td>AB108930</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha Smooth Muscle Actin antibody</td>
			<td>Abcam</td>
			<td>AB124964</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Envision Dual Link System HRP</td>
			<td>Dako</td>
			<td>K4065</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of intact, whole mouse mammary glands for analysis of extracellular matrix expression and gland morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Female mice have 5 pairs of mammary glands. Specifically, there is one pair of cervical glands (#1), two pairs of thoracic glands (#2 and #3), one pair of abdominal glands (#4), and 1 pair of inguinal glands (#5) (Figure 1A). Here, we isolated the #4 glands as they are readily identifiable. In some circumstances, both #4 and #5 glands were isolated together as distinction between the two was difficult. To isolate intact #4 abdominal mammary g...

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

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.

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

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16.1K Views.  University of Nebraska Medical Center. 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. 

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

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

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

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

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

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

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 is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...