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.
