This protocol is the gateway to the extracellular matrix. It's complexity and its structure down to sub micron scale. So the key advantage of this method is that it makes it possible to obtain an extracellular matrix that retains its structural integrity thus opening the way for biochemical and anatomical analysis.
Demonstrating the procedure will be, Alejandro Mayorca Gilani, Raphael Reuten and Maria Rafaeva, currently in my laboratory and Chris Madsen and who was in my laboratory and continued the work at Lund University. Begin by shaving the thorax, abdomen and back of the euthanized mouse with a hair clipper. Disinfect the area with 70%ethanol.
Then pin the mouse to a polystyrene tray, extending its four end hind limbs, as well as its head and tail. Place it under the microsurgery microscope. Using a Mayo straight pattern scissors, make a cutaneous incision running from the sub mandibular region to the lower abdomen and dissect subcutaneously to expose the thoracic wall and peritoneum.
Then use microsurgical scissors to cut the pectoralis major and pectoralis minor muscles along the sixth intercostal space on both sides of the thoracic wall. Cut the sternum along the previous incisions with straight pattern scissors. Then complete a sternotomy by cutting the sternum along its long axis.
Elevate and pin, both sides of the thoracic wall to expose the cardiopulmonary complex. Use round tipped micro forceps to excise the thymus and surrounding adipose tissue by delicately pulling them off their attachments. And cut the esophagus with micro scissors.
Separate the brachiocephalic veins and brachiocephalic artery with sharp micro forceps. Then separate the left common carotid and left subclavian arteries from the underlying tissue to facilitate ligation and cauterization. Use a microneedle holder and sharp micro forceps and a nine oh suture to place stitches above the emergence of the brachiocephalic left common carotid and left subclavian arteries, cauterize the brachiocephalic veins.
Using micro scissors, open an entrance by sectioning the ligament. Introduce a 27 gauge catheter into the trachea and delicately push until trachea branches into the bronchi, taking care to not disrupt the bronchi. Using a six O suture, place three stitches around the trachea to secure the catheter.
Section the mouse at the height of the 12th thoracic vertebra, the descending Aorta runs anteriorly to the spine and should be sectioned here along with the spine. Retrogradely catheterize the Aorta and push the catheter until it reaches the aortic arc. Using a suture, place four stitches around the Aorta, beginning five millimeters below the catheter tip.
Connect the mouse to a pump system using silicone tubing and lower connectors. Perfuse with deionized water at 200 microliters per minute for 15 minutes. Then change the perfusion agent to 0.5%DOC, diluted in deionized water and perfuse overnight.
On the next day, change the perfusion agent to 0.1%SDS diluted in deionized water, and perfuse for eight hours. Then perfuse with deionized water for 24 hours to wash away the SDS and DOC. Resect the decellularized heart and lungs by sectioning its attachments to the thorax.
Store the tissue in a sterile cryo tube in deionized water with 1%penicillin streptomycin and 0.3 micromolar sodium azide at four degrees Celsius. To perform immuno staining, block the sample by incubating it in a cryo tube containing 6%donkey serum and 3%BSA overnight. Then incubate with primary antibody in 3%donkey serum in PBS for 24 hours.
After the incubation wash the sample five times in PBST for one hour per wash. Incubate the sample with fluorescently conjugated secondary antibody in 3%donkey serum in PBS for 24 hours. Then repeat the washes in PBST.
Add the Ionized water and store the sample at four degrees Celsius away from direct light. To image the sample, place it in a glass bottom dish and humidify it with two droplets of storing solution. Set the objective and inspect the sample using fluorescence light.
Switch to computer control, turn on lasers and adjust laser intensity, pinhole aperture, detectors wavelength, gain, resolution and zoom. Set the number and step size for Z stack, then begin acquisition. Excise one lung lobe from a euthanized mouse and place it in a 10 by 10 by five millimeter cryo mold.
Cover it with approximately 500 mile microliters of OCT compound and freeze it on dry ice. Maintain the sample at that temperature. Excise one decellularized lung lobe from a processed mouse, place it in a cryo mold with the largest surface area down and cover it with OCT compound.
Freeze the sample on dry ice and maintain it at that temperature until otherwise required. The sample can be stored for at least 12 weeks. Use a cryostat to section frozen tissue blocks into five micrometer sections at minus 20 degrees Celsius.
And place the sections on adhesive glass slides. Transfer the slides to room temperature until air dried. Briefly immerse the slides in PBS, followed by 4%paraformaldehyde in PBS for 15 minutes.
Wash once in PBS, then twice in distilled water for five minutes per wash. Immerse the slides in Myer's hematoxylin solution for 10 minutes. Next, wash the slides in a Copeland jar under running distilled water for 10 minutes and immerse in eosin solution for seven minutes.
Dip in xylene several times. Apply a few drops of DPX mounting medium and place a glass cover slip. Leave the slides to dry overnight under a chemical hood then scan them in a slide scanner.
After successfully completing the protocol, the heart and lungs and NX tissue will be free of cells. Decellularization can be validated by hematoxylin eosin staining of the ECM scaffolds. The scaffolds retain the dimensions of of fresh organs and its insoliable ECM structure is intact.
ECM scaffolds showed increased permeability and light penetrability. Using this protocol with a motorized microscope stage allows for three dimensional tiled imaging of whole mount samples at sub micron resolution. It is essential to shun flow toward the organs of interest to achieve complete uniform decellurization.
The microsurgical dissection and ligation of vessels must be effective. And at the same time respect the target tissues to maintain native VCM structure. Following this procedure, the scaffolds will be enriched for structural ECM proteins and can be used for mass spectrometry or tissue engineering.
This technique paves the way to high resolution mapping of the extracellular matrix.
