Name: Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands (Video) | JoVE Journal
Uploaded: 2017-05-30T14:00:00.000+00:00
Duration: 11 min 31 s
Description: Learn co-immunoprecipitation and co-immunofluorescence to study protein-protein interactions and co-localization of gap, tight, and adherens junction proteins in murine mammary glands. This cost-effective in vivo method aids breast cancer research.

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5 minutes

I.P. is funded by a Natural Sciences and Engineering Research Council of Canada grant (NSERC #418233-2012); a Fonds de Recherche du Qu&#233;bec-Sant&#233; (FRQS), a Quebec Breast Cancer Foundation career award, and a Leader Founds grant from the Canadian Foundation for Innovation grant. E.D. received a scholarship from the Fondation universitaire Armand-Frappier.

All animal protocols used in this study were approved by the University Animal Care Committee (INRS-Institut Armand-Frappier, Laval, Canada).
1. Identifying Protein Co-localization
<ol>
	<li>From tissue to microscopic slides 
	NOTE: Tissues and sections should be handled on dry ice.
	<ol>
		<li>Excise the mammary glands from an animal (for a complete description of this procedure, refer to Plante et al.)14.</li>
		<li>Embed the excised tissue in freezing/mounting medium on dry ice. Add enough medium to cover the gland. When the medium is solidified, transfer the tissues to a freezer at -80 &#176;C for later use9.</li>
		<li>Using a cryomicrotome set at &#8804; -35 &#176;C, cut the tissues into 7-10 &#956;m-thick sections and place them on microscope slides. 
		NOTE: When possible, place two sections on each slide; the left section will be used as a negative control to verify the specificity of the antibodies and the autofluorescence of the tissue, while the right side will be labeled with the antibodies.</li>
		<li>Keep the sections at -80 &#176;C for later use.</li>
	</ol>
	</li>
	<li>Co-IF staining
	<ol>
		<li>Retrieve the appropriate microscopic slides from the freezer and immediately fix the sections by immersing them in formaldehyde 4% for 15 min at room temperature (RT).</li>
		<li>Then immerse the slides in phosphate-buffered saline (PBS) at RT. Leave the slides in PBS at RT until proceeding to the next step.</li>
		<li>Circle each section of the slide using a commercially available hydrophobic barrier or a water-repellent lab pen (see the Table of Materials). Be careful not to touch the tissue. Immediately add drops of PBS to the tissue and place the slide in a humid histology chamber for the remainder of the procedure. 
		NOTE: The tissue sections must remain moisturized. Alternatively, use a box with a lid and place wet paper towels on the bottom.</li>
		<li>Block each tissue section with 100-200 &#181;L of 3% bovine serum albumin (BSA)-Tris-buffered saline (TBS)-0.1% polysorbate 20 (see the Table of Materials) for 30 min at RT. While the samples are blocking, prepare the primary and secondary antibody solutions by diluting the antibodies in TBS-0.1% polysorbate 20. 
		NOTE: The required concentration for the antibody is provided by the manufacturer; see the Table of Materials and Figures 1 and 2 for examples, as well as Dianati et al.9. Although it is not necessary to work in the dark when using most fluorophore-conjugated antibodies, avoid exposing the antibody solutions or stained tissues to intense, bright light.</li>
		<li>Remove the blocking solution by aspiration and incubate the sections in 100-200 &#181;L of the diluted primary antibody for 60 min at RT. Alternatively, incubate with the primary antibody overnight at 4 &#176;C.</li>
		<li>Remove the primary antibody solution by aspiration and wash the sections with 250-500 &#181;L of TBS-0.1% polysorbate 20 for 5 min. Remove the wash solution by aspiration and repeat the wash twice.</li>
		<li>Remove the wash solution by aspiration and incubate the sections with 100-200 &#181;L of the appropriate fluorophore-conjugated secondary antibody for 60 min at RT.</li>
		<li>Remove the secondary antibody solution by aspiration and wash the sections with 250-500 &#181;L of TBS-0.1% polysorbate 20 for 5 min. Remove the wash solution and repeat twice.</li>
		<li>Repeat step 2.5-2.8 using the appropriate combination of primary and secondary antibodies for the subsequent protein(s) of interest.</li>
		<li>Remove the wash solution by aspiration and perform the nuclei staining by incubating the section with 100-200 &#181;L of 1 mg/mL 4&#39;,6-diamidino-2-phenylindole (DAPI) in TBS-0.1% polysorbate 20 for 5 min at RT.</li>
		<li>Remove the DAPI solution by aspiration and mount the slides using a water-soluble, non-fluorescing mounting medium (see the Table of Materials) and coverslips. Proceed one slide at a time. 
		NOTE: Incubating the nucleus stain for more than 5 min will not change the intensity of the staining. Alternatively, remove the DAPI solution on all slides and incubate the tissues in PBS while mounting the slides.</li>
		<li>Place the slides flat in a 4 &#176;C refrigerator for at least 8 h. Proceed to the fluorescence microscopic imaging (see Figures 1 and 2).</li>
	</ol>
	</li>
	<li>Microscopic imaging
	<ol>
		<li>Visualize the fluorophore-conjugated secondary antibodies using a confocal microscope equipped with the various lasers required to excite the fluorophores at their specific wavelengths. 
		Note: To be able to visualize ducts and alveoli, a 40X objective with a numerical aperture of 0.95 is suggested. An example of the specific settings is provided in Figure 1.</li>
		<li>Verify the localization of each protein individually by scanning the image one wavelength at the time. 
		Note: At this stage, it is important to critically analyze the localization of the junctional proteins. To be able to form junctional nexuses, these proteins must be localized at the plasma membrane.</li>
		<li>Determine the co-localization of proteins by merging the images scanned with the lasers at the different wavelengths. 
		Note: Protein co-localization can be visualized by the change of color resulting from the emission of two or more fluorophores at the same location and can be measured using the appropriate software (Figures 1 and 2; also see Reference 9).</li>
	</ol>
	</li>
</ol>
2. Studying PPIs
NOTE: Abdominal mammary glands should be used to study PPIs, as thoracic glands are in close association with the pectoral muscles. Excise the mammary glands (for a complete description of this procedure, refer to Plante et al.)14 and keep them at -80 &#176;C for later use.
<ol>
	<li>Lysate preparation
	<ol>
		<li>Place weighing paper and 2 mL microcentrifuge tubes on dry ice to pre-cool them before proceeding with the next steps.</li>
		<li>Take the mammary gland tissue from -80 &#176;C and keep them on dry ice.</li>
		<li>Weigh the tissues on the pre-chilled weighing papers and then transfer the tissue to 2 mL microcentrifuge tubes (handle on dry ice). Use between 50 and 100 mg of tissue per sample. Keep the tissue on dry ice until step 2.1.5.</li>
		<li>Prepare the required amount of triple detergent lysis buffer supplemented with NaF, NaVO3, and protease/phosphatase inhibitor, as indicated in the Table of Materials, using the following formulas. Mice: required buffer (&#181;L) = mouse tissue weight (mg) x 3; Rat: required buffer (&#181;L) = rat tissue weight (mg) x 5.</li>
		<li>Add the required amount of ice-cold lysis buffer (calculated in step 2.1.4) to the 2 mL tube containing the tissue. 
		NOTE: In steps 2.1.5-2.1.6, proceed with one single tube at a time.</li>
		<li>Homogenize the tissue for 30-40 s using continuous homogenization on a tissue grinder; always keep the tube on ice. Adjust the tissue homogenizer to medium speed and gently move the grinder up and down inside the tube.</li>
		<li>Repeat steps 1.6 and 1.7 with the other tubes.</li>
		<li>Incubate the lysates on ice for 10-30 min.</li>
		<li>Centrifuge the tubes at 170 x g for 10 min at 4 &#176;C.</li>
		<li>Meanwhile, identify 6-10 microcentrifuge tubes (0.6 mL) for each sample and keep them on ice.</li>
		<li>Once the centrifugation is done, check the tubes. Ensure that they contain a top layer of fat, clear, yellow-to-pink lysates (depending on the stage of development) and a pellet.</li>
		<li>Create a hole in the lipid layer using a 200-&#181;L pipette tip to access the liquid phase. Change the tip and collect the lysate without disturbing the pellet or aspirating the lipid layer. Aliquot the lysate in pre-labeled tubes on ice (step 2.1.11) and store them at -80 &#176;C.</li>
		<li>Use an aliquot to quantify the protein concentration using an appropriate commercially available kit (see the Table of Materials).</li>
	</ol>
	</li>
	<li>Indirect immunoprecipitation
	<ol>
		<li>On ice, thaw two aliquots of the total mammary gland lysates prepared previously. 
		NOTE: One aliquot will be used for the IP of the target protein, while the other will serve as the negative control.</li>
		<li>Collect 500-1,000 &#181;g of the lysate and dilute it in PBS to reach a final volume of 200 &#181;L in each 1.5 mL tube. 
		NOTE: The amount of lysate to be used depends on the abundance of the protein of interest and the efficiency of the antibody (see Figure 3 for an example, as well as the Table of Materials). To optimize for each target, different amounts of lysate (i.e., 500, 750, and 1,000 &#181;g) and antibody (i.e., 5, 10, and 20 &#181;g) should be used. Proceed with the following steps (2.2.3-2.3.7.4).</li>
		<li>Add the antibody against the antigen of interest to the first tube of lysate and keep it on ice. 
		NOTE: The required amount is usually suggested on the instruction sheet provided by each company (see the Table of Materials).</li>
		<li>In the second tube, prepare a negative control by adding the same concentration of isotype IgG control as the antibody used in step 2.2.3.</li>
		<li>Incubate the tubes overnight at 4 &#176;C on a tube roller-mixer at low speed.</li>
		<li>The following day, add 50 &#181;L of magnetic beads to new 1.5 mL tubes for pre-washing.
		<ol>
			<li>Select either Protein A or Protein G magnetic beads based on the relative affinity to the antibody.</li>
			<li>It is important to avoid using aggregated beads; gently mix the bead suspension until it is uniformly re-suspended before adding it to the tubes.</li>
		</ol>
		</li>
		<li>Place the tubes containing the beads on the magnetic stand and allow the beads to migrate to the magnet. Remove the storage buffer from the beads using a 200 &#181;L pipette.</li>
		<li>Wash the beads by adding 500 &#181;L of PBS-0.1% polysorbate 20 and vortex the tubes vigorously for 10 s.</li>
		<li>Put the tubes back on to the magnetic stand and allow the beads to migrate to the magnet.</li>
		<li>Remove the excess wash buffer by pipetting with a 200 &#181;L pipette.</li>
		<li>Add the reaction complex (lysate-antibody) from step 2.2.5 to the beads and incubate for 90 min at RT on the roller mixer.</li>
		<li>Place the tubes on the magnetic stand and allow the beads to migrate to the magnet. Using a 200 &#181;L pipette, aspirate and discard the lysate and place the tubes on ice.</li>
		<li>Wash the beads by adding 500 &#181;L of PBS, placing the tubes on the magnetic stand, and removing the liquid using a 200 &#181;L pipette. Repeat this wash step. During the wash steps, avoid vortexing and keep the samples on ice.</li>
		<li>Wash the beads once with PBS-0.1% polysorbate 20 without vortexing and discard the last wash buffer using a 200 &#181;L pipette tip.</li>
		<li>To elute, add 20 &#181;L of 0.2 M acidic glycine (pH = 2.5) to the tubes and shake them for 7 min on the roller mixer.</li>
		<li>Centrifuge at high speed for a few seconds (quick spin) and collect the supernatant in a fresh ice-cold tube.</li>
		<li>Repeat steps 2.2.14 and 2.2.15 for each tube. 
		NOTE: The final volume will be 40 &#181;L.</li>
		<li>Add 10 &#181;L of 4x Laemmli buffer to the 40-&#181;L eluted sample from step 2.2.16. 
		NOTE: The color will turn yellow due to the acidic pH.</li>
		<li>Immediately add 1 M Tris (pH = 8), one drop at a time, to the eluted sample from step 2.2.18 until its color turns blue. Proceed to the next tubes.</li>
		<li>Boil the samples from step 2.2.18 at 70-90 &#176;C for 10 min. Proceed immediately to gel electrophoresis. Alternatively, transfer the samples to a freezer at -80 &#176;C until loading.</li>
	</ol>
	</li>
	<li>Downstream application: gel electrophoresis followed by Western blot
	<ol>
		<li>Prepare separating and stacking SDS-PAGE acrylamide gels (1.5 mm thickness) following standard procedures15. 
		NOTE: The choice of gel (8-15% acrylamide, gradient: see the Table of Materials) should be determined based on the molecular size of the protein to be precipitated and of the potential binding partners to be analyzed. These proteins must be resolved from each other to allow for proper immunodetection.</li>
		<li>Thaw the immunoprecipitation (IP)-eluted samples (step 2.2.20) on ice.</li>
		<li>Prepare protein lysates from the same samples (used for the IP procedure above). Use 50 &#181;g of total lysate and add 4X Laemmli sample buffer. Boil the samples at 70-90 &#176;C for 5 min and place on ice until loading. 
		NOTE: These samples will be loaded beside the eluted IP sample to demonstrate the presence of precipitated proteins in the total lysate.</li>
		<li>Load the prepared lysates from step 2.3.3 and the precipitated samples from step 2.2.20 side-by-side in an acrylamide gel and run them in running buffer (10x running buffer: 30.3 g of Tris, 144.1 g of glycine, and 10 g of SDS in 1 L of distilled water) at 100 V for approximatively 95 min, or until the edge of the migrating proteins reaches the bottom of the gel.</li>
		<li>Transfer the gels to a nitrocellulose or PVDF membrane using a standard protocol9,15.</li>
		<li>Block the membrane for 1 h on a rocker on low speed in 5% dry milk-TTBS (20 mM Tris, 500 mM NaCl, and 0.05% polysorbate 20).</li>
		<li>Identify whether the precipitation was successful.
		<ol>
			<li>Probe the membrane using the first antibody against the precipitated protein, diluted in 5% dry-milk-TTBS at the concentration recommended by the manufacturer, overnight at 4 &#176;C on a rocking platform with slow agitation. 
			NOTE: See the Table of Materials for recommendations.</li>
			<li>The following day, wash the membrane 3 times for 5 min each with TTBS on a rocking platform with high agitation.</li>
			<li>Incubate the membrane in the appropriate secondary antibody conjugated with horseradish peroxidase (HRP), diluted in TTBS, for 1 h at RT on a rocking platform with slow agitation. 
			NOTE: Alternatively, a secondary antibody conjugated with a fluorochrome can be used if an appropriate apparatus to detect the signal is available.</li>
			<li>Perform 3 to 6 washes, each for 5 min, with TTBS on a rocking platform with high agitation. Analyze the signal of the secondary antibody by incubating the membrane with a commercially available luminol solution (see the Table of Materials) and follow the manufacturer instructions. Detect the signal using a chemiluminescence imaging system (see the Table of Materials). 
			NOTE: For a detailed protocol on Western blot analysis, see Reference 16.</li>
		</ol>
		</li>
		<li>To identify interacting proteins, perform steps 2.3.7.1-2.3.7.4 using the appropriate antibodies on the same blot. 
		NOTE: If proteins are interacting, the binding partners will be co-immunoprecipitated with the target protein and will thus be detectable by Western blotting. Step 2.3.8 can be repeated with more antibodies to determine whether other proteins reside in the same proteins complex, as long as the molecular weights of the proteins differ enough to be well-separated on the gel and membrane.</li>
		<li>To confirm that the identified binding partners are not artifacts, reciprocal IP should be performed. 
		NOTE: This is performed by repeating steps 2.2.1-2.2.20 with the same lysate but precipitating one of the binding partners identified in step 3.8. Then, steps 3.1-3.8 are repeated using the primary antibody against the first protein of interest.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to declare.

Cell-cell interactions via junctions are required for the proper function and development of many organs, such as the mammary gland. Studies have shown that junctional proteins can regulate the function and stability of one another and activate signal transduction by tethering each other at the cell membrane10. The protocols presented in the current manuscript have provided interesting findings about junctional protein differential expression, localization, and interaction during normal murine gla...

Mammary gland growth and development occurs mainly after birth. This organ constantly remodels itself throughout the reproductive life of a mammal1. The adult mammary gland epithelium is comprised of an inner layer of luminal epithelial cells and an outer layer of basal cells, mainly composed of myoepithelial cells, surrounded by a basement membrane2. For a good review on mammary gland structure and development, the reader can refer to Sternlicht1. Cell-cell interactions via gap (GJ), tight (TJ), and adherens (AJ) junctions are necessary for the normal development and function of the gland1,3,4,5,6. The main components of these junctions in the murine mammary gland are Cx26, Cx30, Cx32, and Cx43 (GJ); Claudin-1, -3, -4, and -7 and ZO-1 (TJ); and E-cadherin, P-cadherin, and &#946;-catenin (AJ)7,8. The levels of expression of these different junctional proteins vary in a stage-dependent manner during mammary gland development, suggesting differential cell-cell interaction requirements9. GJ, TJ, and AJ are linked structurally and functionally and tether other structural or regulatory proteins to the neighboring sites of adjacent cells, thus creating a junctional nexus10. The composition of the junctional nexus can impact bridging with the underlying cytoskeleton, as well as nexus permeability and stability, and can consequently influence the function of the gland8,9,10,11. The components of intercellular junctions residing in junctional nexuses or interacting with one another at different developmental stages of mammary gland development were analyzed recently using co-immunofluorescence (co-IF) and co-immunoprecipitation (co-IP)9. While other techniques allow for the evaluation of the functional association between proteins, these methods are not presented in this manuscript.
As proteins merely act alone to function, studying protein-protein interactions (PPIs), such as signal transductions and biochemical cascades, is essential to many researchers and can provide significant information about the function of proteins. Co-IF and microscopic analysis help to evaluate a few proteins that share the same subcellular space. However, the number of targets is limited by the antibodies, which must be raised in different animals, and by the access to a confocal microscope equipped with different wavelength lasers and a spectral detector for multiplexing. Co-IP confirms or reveals high-affinity physical interactions between two or more proteins residing within a protein complex. Despite the development of novel techniques, such as fluorescence resonance energy transfer (FRET)12 and proximity ligation assay (PLA)13, which can simultaneously detect the localization and interactions of proteins, co-IP remains an appropriate and affordable technique to study interactions between endogenous proteins.
The step-by-step method described in this manuscript will facilitate the study of protein localization and PPIs and point out pitfalls to avoid when studying endogenous PPIs in the mammary glands. The methodology starts with the presentation of the different preservation procedures for the tissues required for each technique. Part 1 presents how to study protein co-localization in three steps: i) the sectioning of mammary glands, ii) the double- or triple-labeling of different proteins using the co-IF technique, and iii) the imaging of protein localization. Part 2 shows how to precipitate an endogenous protein and identify its interacting proteins in three steps: i) lysate preparation, ii) indirect protein immunoprecipitation, and iii) binding partner identification by SDS-PAGE and Western blot. Each step of this protocol is optimized for rodent mammary gland tissues and generates high-quality, specific, and reproducible results. This protocol can also be used as a starting point for PPI studies in other tissues or cell lines.

<table><tbody><tr><td>Mice strain and stage</td><td>St. Constant, Quebec, Canada</td><td></td><td>C57BL/6 Femals; pregnancy day 18 (P18) and lactation day 14 (L14), Charles River Canada&amp;nbsp;</td></tr><tr><td>PBS 10x (stock)</td><td></td><td></td><td>1) Dissolve 80 g NaCl (F.W.: 58.44), 2 g KCl (F.W. 74.55), 26.8 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;#183;7H&lt;sub&gt;2&lt;/sub&gt;O (F.W. 268.07) and 2.4 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (F.W.:136.09) in 800 mL distilled water;&lt;br/&gt; 2) Adjust the PH to 7.4;&lt;br/&gt; 3) Add water to reach to the 1 L final volume.</td></tr><tr><td>TBS 10x (stock)</td><td></td><td></td><td>1) Dissolve 60.5 g TRIS, 87.6 g NaCl in 800 mL distilled water;&lt;br/&gt;2) Adjust the pH to 7.5;&lt;br /&gt;3) Add water to reach to the 1 L final volume.</td></tr><tr><td colspan="4">&lt;strong&gt;Part 1: Immunofluorescence&lt;/strong&gt;</td></tr><tr><td>Freezing media</td><td>VWR International, Ville Mont-Royal, QC, Canada</td><td>95067-840</td><td>VMR frozen sections compound&amp;nbsp;</td></tr><tr><td>Microtome</td><td>Mississauga, ON, Canada</td><td>956640</td><td>Microm HM525, Thermo fisher scientific HM525 NX Cryostat 115 V 60 Hz</td></tr><tr><td>Blades</td><td>C.L. Sturkey, Inc. Les Produits&amp;nbsp;Scientifiques&amp;nbsp;ESBE&amp;nbsp;St-Laurent, QC, Canada</td><td>BLM1001C</td><td>High profile gold coated blades</td></tr><tr><td>Pap pen</td><td>Cedarlane, Burlington, ON, Canada</td><td>8899</td><td>Super PAP Pen, Thermo fisher scientific</td></tr><tr><td>Microscopic slides</td><td>Fisher Scientific, Burlington, ON, Canada</td><td>12-550-15</td><td>Fisherbrand&amp;nbsp;Superfrost Plus Microscope Slides</td></tr><tr><td>Formaldehyde</td><td>BioShop Canada Inc, Burlington, ON, Canada</td><td>FOR201.1</td><td>Forlmadehyde</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Santa Cruz Biotechnology, Inc, California, USA</td><td></td><td></td></tr><tr><td>Blocking solution</td><td></td><td></td><td>3% BSA in TBS</td></tr><tr><td>Wash solution</td><td></td><td></td><td>TBS-Tween 20 0.1%</td></tr><tr><td>Polysorbate 20</td><td>Oakville, ON, Canada</td><td>P 9416</td><td>Tween 20, Sigma-Aldrich</td></tr><tr><td>Mounting media</td><td>Cedarlane, Burlington, ON</td><td>17984-25(EM)</td><td>Fluoromount-G</td></tr><tr><td>First &amp;amp; secondary antibodies</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>E-Cadherin (4A2) Mouse mAb (#14472s) 1/50 (Cell Signaling) with anti-mouse IgG Fab2 Alexa Fluor 555 (#4409s), Cell Signaling&amp;nbsp;</td></tr><tr><td>First &amp;amp; secondary antibodies&amp;nbsp;</td><td>Life technologies, Waltham, MA, USA &amp;amp; Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>Claudin-7 (#34-9100) 1/100 (Life Technologies) with anti-rabbit IgG Fab2 Alexa Fluor 488 (#4412s) (Cell Signaling)&amp;nbsp;</td></tr><tr><td>First &amp;amp; secondary antibodies&amp;nbsp;</td><td>Santa Cruz Biotechnology, Inc, California, USA; Fischer Scientific, Burlington, ON, Canada&amp;nbsp;</td><td>See Comments</td><td>&amp;beta;-Catenin Antibody (C-18): sc-1496 (SANTA CRUZ) with anti-Goat IgG (H+L) Alexa Fluor 568 (#A11057), Molecular Probe (Fisher Scientific)</td></tr><tr><td>First &amp;amp; secondary antibodies&amp;nbsp;</td><td>Life technologies, Waltham, MA, USA &amp;amp; Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>Connexin26&amp;nbsp; (#33-5800) 1/75 (Life Technologies) with anti-mouse IgG Fab2 Alexa Fluor 647 (#4410s)&amp;nbsp;</td></tr><tr><td>Nuclei stain</td><td>Fisher Scientific, Burlington, ON, Canada</td><td>D1306</td><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride) 1/1,000 in PBS</td></tr><tr><td>Fluorescent microscope</td><td>Nikon Canada, Mississauga, On, Canada</td><td></td><td>Nikon A1R+ confocal microscopic laser equipped with a spectral detector&amp;nbsp;</td></tr><tr><td>Software of IF images analysis</td><td>Nikon Canada, Mississauga, On, Canada</td><td></td><td>NIS-elements software (version 4)</td></tr><tr><td colspan="4">&lt;strong&gt;Part 2: Immunoprecipitation&lt;/strong&gt;</td></tr><tr><td>Triple-detergent Lysis buffer (100&amp;nbsp;mL) pH=8.0</td><td></td><td></td><td>1) Mix 50 mM TRIS (F.W.: 121.14), 150&amp;nbsp;mM NaCl (F.W.: 58.44), 0.02% Sodium Azide, 0.1% SDS, 1% NONIdET P40, 0.5% Sodium Deoxycholate in 80 mL distilled H&lt;sub&gt;2&lt;/sub&gt;O.&lt;br/&gt; 2) Adjust the pH to 8.0 with HCl 6&amp;nbsp;N (~0.5&amp;nbsp;mL).&lt;br/&gt; 3) Adjust the volume to 100&amp;nbsp;mL. Keep it in fridge.&lt;br/&gt; At the day of protein extraction, use 1/100 NaVo&lt;sub&gt;3&lt;/sub&gt;, 1/100 protease/phosphatase inhibitor and 1/25 NAF in calculated amount of Triple detergent lysis buffer:&lt;br/&gt; Sodium Fluoride (stock) solution 1.25&amp;nbsp;M (F.W.: 41.98), Sodium Orthovanadate (stock) Solution 1&amp;nbsp;M (F.W.: 183.9)</td></tr><tr><td>Protease/phosphatase inhibitor</td><td>Fisher Scientific, Burlington, ON</td><td>78441</td><td>Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100x)</td></tr><tr><td>Protein dosage</td><td>Thermo Scientific, Rockford, Illinois, USA</td><td>23225</td><td>Pierce BCA protein assay kit&amp;nbsp;</td></tr><tr><td>Tissue grinder</td><td>Fisher Scientific, Burlington, ON</td><td>FTH-115</td><td>Power 125, Model FTH-115</td></tr><tr><td>Magnetic beads and stand</td><td>Millipore, Etobicoke, ON, Canada</td><td></td><td>PureProteome Protein G Magnetic Bead System (LSKMAGG02)</td></tr><tr><td>Wash solution for IP</td><td></td><td></td><td>PBS or PBS-Tween20 0.1% depending to the step</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>IgG Rabbit (rabbit (DA1E) mAb IgG Isotype control (#3900s) (Cell Signaling) 0.5 &amp;micro;L/200 &amp;micro;L</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>IgG Mouse mouse (G3A1) mAb IgG Isotype control (#5415s) (Cell Signaling) 0.5 &amp;micro;L/200 &amp;micro;L</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Sigma-Aldrich, Oakville, ON, Canada</td><td>See Comments</td><td>Connexin43 (#C6219) (Sigma-Aldrich) 4&amp;nbsp;&amp;micro;L/200&amp;nbsp;&amp;micro;L</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>E-cadherin (4A2) Mouse mAb (#14472s) (Cell Signaling) 1 &amp;micro;L/200 &amp;micro;L</td></tr><tr><td>Laemmli buffer&amp;nbsp;</td><td>BIO-RAD, Mississauga, Ontario, Canada</td><td>1610747</td><td>4x Laemmli Sample Buffer (Add &amp;beta;-mercaptoethanol following manufacturer recommendation)</td></tr><tr><td>Acidic glycine&amp;nbsp;</td><td>Fisher Scientific, Burlington, ON</td><td>PB381-5</td><td>0.2 M glycine; adjust pH=2.5 with HCl&amp;nbsp;</td></tr><tr><td>Tris&amp;nbsp;</td><td>Fisher Scientific, Burlington, ON</td><td>BP152-1</td><td>1 M (pH=8)&amp;nbsp;</td></tr><tr><td>SDS-PAGE acrylamide gels&amp;nbsp;</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1610180 -5</td><td>TGX Stain-Free FastCast Acrylamide Solutionss (7.8%, 10%, 12%)</td></tr><tr><td>Running buffer 10x&amp;nbsp;</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1704272</td><td>Tris 30.3&amp;nbsp;g/glycine 144.1&amp;nbsp;g /SDS 10&amp;nbsp;g in 1 L distilled water</td></tr><tr><td>Membranes</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1704272</td><td>PVDF membranes, Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td></tr><tr><td>Transfer method</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1704155</td><td>Trans-Blot Turbo Transfer System</td></tr><tr><td>Dry Milk</td><td>Smucker Food of Canada Co, Markham, ON, Canada</td><td></td><td>Fat Free Instant Skim Milk Powder, Carnation</td></tr><tr><td>Blocking solution for blots</td><td></td><td></td><td>5% dry milk in TBS-Tween 20 0.1%</td></tr><tr><td>Washing solutions for blots</td><td></td><td></td><td>TBS-Tween 20 0.1%</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Sigma-Aldrich, Oakville, Ontario &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>Connexin43 (#C6219) (Sigma-Aldrich) 1/2,500 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Cell Signaling, Beverly, MA, USA &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>E-cadherin (24E10) rabbit mAb 1/1,000 (#3195s) (Cell Signaling) 1/1,000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Life technologies, Waltham, MA, USA &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>Claudin-7 (#34-9100) (Life technologies) 1/1,000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Life technologies, Waltham, MA, USA &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>Claudin3 (#34-1700) (Life technologies) 1/1,000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Luminol solution for signal detection on blots</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1705061</td><td>Clarity Western ECL Blotting Substrate</td></tr><tr><td>Imaging blots</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1708280</td><td>ChemiDoc MP imaging system</td></tr><tr><td>Analayzing blots</td><td>BIO-RAD, Mississauga, ON, Canada</td><td></td><td>ImageLab 5.2 software&amp;nbsp;</td></tr></tbody></table>

analysis of protein-protein interactions and co-localization between components of gap, tight, and adherens junctions in murine mammary glands

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

To determine whether GJ, AJ, and TJ components can interact together in the mammary gland, co-IF assays were first performed. Cx26, a GJ protein, and β-Catenin, an AJ protein, were probed with specific antibodies and revealed using fluorophore-conjugated mouse-647 (green, pseudocolor) and goat-568 (red) antibodies, respectively (Figure 1B and C). Data showed that they co-localize at the cell membrane of epithelial cells in the mice mammary gland on lactat...

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

The overall goal of this method is to determine whether two or more intercellular junction proteins co-localize within cells and to further assess their physical interaction via co-immunoprecipitation. This method can help to answer key questions in the biology of the mammary gland field including the roles played by junctions in normal development and diseases such as breast cancer. The main advantage of this technique is that it can provide significant information about protein-protein interaction in vivo at a relatively low cost.To perform co-immuno fluorescent staining, retrieve the appropriate microscopic slides from the freezer. Immediately fix the sections by immersing them in four percent formaldehyde for 15 minutes at room temperature. Then, immerse the slides in phosphate-buffered saline at room temperature.Leave the slides there until proceeding to the next step. Circle each section of the slide using a commercially-available hydrophobic barrier or a water-repellent lab pen. Immediately add drops of PBS to the tissue, and place the slide in a human histology chamber for the remainder of the procedure.Block each tissue section with 100 to 200 microliters of three percent Bovine Serum Albumin-Tris-Buffered Saline, 0.1%polysorbate-20, for 30 minutes at room temperature. While the samples are blocking, prepare the primary and secondary antibody solutions by diluting the antibodies in TBS 0.1%polysorbate-20. After removing the blocking solution by aspiration, incubate the sections in 100 to 200 microliters of the diluted primary antibody for 60 minutes at room temperature.Then remove the primary antibody solution by aspiration and wash the sections with 250 to 500 microliters of TBS 0.1%polysorbate-20 for five minutes. Remove the wash solution by aspiration before repeating the wash twice. Aspirate the wash solution and incubate the sections with 100 to 200 microliters of the appropriate Fluoro-4 conjugated secondary antibody for 60 minutes at room temperature.After removing the secondary antibody solution by aspiration, wash the sections with 250 to 500 microliters of TBS 0.1%polysorbate-20 for five minutes. Remove the wash solution and repeat twice. Repeat these steps using the appropriate combination of primary and secondary bodies for any subsequent proteins of interest.Following removal of the final wash, perform the nuclei staining by incubating the section with 100 to 200 microliters of one milligram per millileter DAPI and TBS 0.1%polysorbate-20 for five minutes at room temperature. Aspirate the DAPI solution and mount the slides using a water-soluble, non-fluorescing mounting medium and cover slips. Place the slides flat in a four-degree Celsius refrigerator for at least eight hours.Proceed to the fluorescence microscopic imaging as described in the text protocol. Collect 500 to 1000 micrograms of the lysate and dilute it in PBS to each a final volume of 200 microliters in each 1.5-milliliter tube. Add the antibody against the antigen of interest to the first tube of lysate, and keep it on ice.Incubate the tubes overnight at four degrees Celsius on a tube-roller mixer at low speed. The following day, add 50 microliters of magnetic beads to new 1.5-milliliter tubes for pre-washing. Place the tubes containing the beads on the magnetic stand and allow the beads to migrate towards the magnet.Remove the storage buffer from the beads using a 200-microliter pipette. Wash the beads by adding 500 microliters of PBS 0.1%polysorbate-20, and vortex the tubes vigorously for 10 seconds. Then, put the tubes back onto the magnetic stand and allow the beads to migrate towards the magnet.Remove the excess wash buffer by pipetting with a 200-microliter pipette. Add the lysate antibody reaction complex to the beads and incubate for 90 minutes at room temperature on the roller mixer. Following the incubation, place the tubes on the magnetic stand and allow the beads to migrate toward the magnet.Using a 200-microliter pipette, aspirate and discard the lysate and place the tubes on ice. Wash the beads twice by adding 500 microliters of PBS, placing the tubes on the magnetic stand, and removing the liquid as before. During the wash steps, avoid vortexing, and keep the samples on ice.Wash the beads once with PBS 0.1%polysorbate-20 without vortexing and discard the last wash buffer using a 200-microliter pipette tip. To elute, add 20 microliters of 0.2 molar acidic glycine to the tubes and shake them for seven minutes on the roller mixer. After centrifuging at high speed for a few seconds, collect the supernatant in a fresh, ice-cold tube.Repeat the wash and elution steps for each tube. Then, add 10 microliters of 4x buffer to the 40-microliter eluted sample. Immediately add one molar Tris to the eluted samples, one drop at a time, until its color turns blue, and then proceed to the next tubes.Boil the eluted samples at 70 to 90 degrees Celsius for 10 minutes. Load the prepared lysates and the precipitated samples side-by-side in an SDS-PAGE acrylamide gel. Run the gel in running buffer at 100 volts for approximately 95 minutes, or until the edge of the migrating proteins reaches the bottom of the gel.Subsequently, transfer the gels to a nitrocellulose or PVDF membrane using a standard protocol. Then, block the membrane for one hour on a rocker on low speed in five-percent dry milk TTBS. To identify whether the precipitation was successful, probe the membrane using the first antibody against the precipitated protein.Dilute it in five percent dry milk TTBS at the concentration recommended by the manufacturer. Incubate the membrane overnight at four degrees Celsius on a rocking platform with slow agitation. The following day, wash the membrane three times for five minutes each with TTBS on a rocking platform with high agitation.Then, incubate the membrane in the appropriate secondary antibody conjugated with horseradish peroxidase, diluted in TTBS, for one hour at room temperature on a rocking platform with slow agitation. Perform three to six washes, each for five minutes, with TTBS on a rocking platform with high agitation. Analyze the signal of the secondary antibody by incubating the membrane with a commercially-available luminol solution and follow the manufacturer's instructions.Detect the signal using a chemiluminescence imaging system. To identify interacting proteins, repeat these steps using the appropriate antibodies on the same blot. To determine whether gap junction, adherence junction, and tight junction components can interact together in the mammary gland, co-immunofluorescence assays were performed.The co-immunofluorescence assay shows that beta-catenin, an integral protein of adherens junction, and Connexin 26, a gap junction protein, co-localize at the cell membrane in mice mammary glands on lactation day seven, as reflected by the yellow color. Cell nuclei are visualized with blue DAPI staining. Cryosections from mammary glands on pregnancy day 18 were cut and immunofluorescently stained for Claudin 7, a tight junction protein shown in green, E-Cadherin, an adherens junction protein shown in red, and Connexin 26, a gap junction protein shown in coral blue.Nuclei were stained with blue DAPI. E-Cadherin and Claudin 7 colocalization is displayed as a yellow to light orange color, while Connexin 26 colocalization with E-Cadherin and Claudin 7 resulted in white-punctuated staining. To find out which junctional proteins intermingle and physically tether together at the cell membrane, co-immunoprecipitation was performed using mammary gland tissues from lactating mice.Results showed that Connexin 43, a component of the gap junction, interacts with E-Cadherin and Claudin 7, but not with Claudin 3. These results were confirmed by the reciprocal immunoprecipitation. When E-Cadherin was immunoprecipitated, it interacted with Connexin 43 and Claudin 7.Once mastered, this technique can be done in two days for co-immunofluorescence for four to eight slides, and slides can be imaged the following day if it is performed properly. While attempting the co-immunofluorescence procedure, it is important to remember to choose different Fluoro 4 conjugated antibodies raised in different spaces with non-overlapping wavelengths and to select the proper protein A or G magnetic beads compatible with the antibodies used for co-immunoprecipitation. After its development, this technique paved the way for researchers in the field of cell biology to explore intercellular junctions'interplay in murine mammary glands.Don't forget that working is extremely hazardous and precautions should be always taken while performing this procedure.

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Research

JoVE Journal

Developmental Biology

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

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.

Evaluation of Mammary Gland Development and Function in Mouse Models

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.

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

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

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

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

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

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

Intravital Subcellular Microscopy of the Mammary Gland

Learn co-immunoprecipitation and co-immunofluorescence to study protein-protein interactions and co-localization of gap, tight, and adherens junction proteins in murine mammary glands. This cost-effective in vivo method aids breast cancer research.

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands (Video) | JoVE Journal

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

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