The overall goal of this procedure is to characterize and quantify atherosclerotic lesions in the MI aortic sinus. This is accomplished by first dissecting an extracted heart in order to separate the apical portion and locate the aortic sinus. Next paraffin blocks are prepared with the processed apical portion of the heart.
Then the aortic sinus is sectioned and mounted onto slides. Finally, the tissue sections are stained. Ultimately, results can be obtained that show different sizes and compositions of atherosclerotic lesions through histochemical and immunohistochemical analysis.
Demonstrating the procedure will be Daniel Venga penal, a graduate student from my laboratory. The McMaster University Animal Research Ethics Board has pre-approved all procedures described herein after anesthetizing an LDLR double negative mouse and extracting the blood euthanize and fix it before isolating the heart according to the text protocol, use a scalpel to make a transversal cut on the heart perpendicular to the ascending aorta. Place the apical or top portion of the heart in an embedding cassette and submerge it in Formin.
Then place the cassette into a tissue processor and process overnight. Using the program outlined in the text protocol for the histology molds melt paraffin wax overnight at 62 degrees Celsius. Place each process tissue into a deep histology mold, ensuring that the interface of the heart is touching the base of the mold, and then pour melted paraffin wax to completely fill the mold.
Use a labeled plastic cassette to cover the mold. After cooling the molds on ice or a cold surface, separate the block from the mold to section the aorta. Position the block in the specimen holder of the microtome so that it can be sectioned from the inside toward the top of the heart.
Adjust the microtome to 10 microns and section the heart. Collect sections on a glass slide and examine under a light microscope to determine position and orientation. The sinus should appear as three bipartite valve bases with attached leaflets.
When one or two valves of the aortic sinus become evident, adjust the angle of the block as necessary. When the valves are reached, adjust the microtome to cut it four to five microns. Mounting the first 10 sections each at the top of individual glass slides, and then mounting each subsequent group of 10 sections on the next position.
On the slides. Continue to collect sections until no atherosclerotic lesion is observed. To stain sections with hematin and eoin to visualize lesions, put the slides in an air dryer for eight minutes.
Then de parize the sections by using xylene to wash them four times for three minutes each. Wash the slides in 100%ethanol three times for three minutes each. Then 70%ethanol and 50%ethanol.
Three times for three minutes each. Use distilled water to rinse the slides and then immerse them in. MA's hematin for 15 minutes.
Wash and running. Tap water for five minutes. Then wash and distilled water for five minutes after immersing in eoin Y for one to five minutes.
Wash and distilled water three times for five minutes each. 50%ethanol two times for two minutes each. Then 70%ethanol three times for two minutes each wash, and 100%ethanol.
Three times for two minutes each. Then wash and xylene four times for two minutes each. Use a xylene based mounting medium to mount the sections and capture images under a light microscope.
Use imaging software to identify and quantify lesions, which can be determined at specific distances from the aortic sinus at 40 to 50 micron intervals, depending on the thickness of the sections, refer to the text protocol for immunohistochemical and immunofluorescence staining. Five week old LDLR. Positive mice were fed a standard diet or a high fat diet for 10 weeks before their hearts were fixed, isolated, and sectioned as shown here.
When mice are fed a standard chow diet, atherosclerotic development is very limited and may not be detectable at 15 weeks of eight. A high fat diet significantly accelerates atherogenesis in this model and induces the formation of large advanced lesions containing ne chronic acellular cores and fibrous caps. Atherosclerotic lesions can be further characterized by staining for the presence of specific cell types and factors that define stages of lesion development.
Monocyte infiltration into the intima represents one of the earliest events of atherogenesis intima. Monocytes differentiate into macrophages that engulf cellular debris and lipids. Lipid engorged macrophages known as foam cells create a fatty streak in the artery wall seen here.
Macrophage foam cells can be detected at all stages of lesion development by staining with antibodies against specific macrophage markers such as F four 80 and MAC three. Vascular smooth muscle cells or v SMCs are confined to the medial layer of a healthy artery. VSCs are induced to migrate and proliferate in the intima during the development of an advanced lesion.
As can be seen in this panel, V SMCs can be identified by staining with an antibody against alpha actin After its development. This technique paid the way for researchers in the field of cardiovascular disease to explore the development and progression of atherosclerotic lesions in rods.
