High-fat diet-fed C57BL/6 mouse

小鼠经口灌胃葡萄糖负荷的耐受试验。禁食 4–6 小时；普通饮食按 1 g/kg、高脂饮食 4 周以上按 0.5 g/kg 给予 50% 葡萄糖溶液；负荷前及负荷后 15、30、60、90、120 分钟测定血糖。不同组别与样本量按具体研究设计确定，本方案不预设实验分组。

获得可用于主动脉根及头臂动脉病变面积和组成定量的固定或冷冻组织块及切片。

获得主动脉根和头臂动脉切片中油红 O 阳性病变面积，并按切片或血管截面面积归一化。

获得可用于兔主动脉斑块组织学分级和组成的 HE 染色切片。

基线与负荷后 15、30、60、90、120 分钟血糖时间序列；可计算血糖曲线下面积。血浆胰岛素结果按所用 ELISA 的校准与量程判读。

获得可供光学显微镜观察的 H&E 染色组织切片：细胞核蓝紫色、细胞质与结缔组织纤维粉红色，用于观察组织中细胞的整体结构与形态细节。本方案本身不产出病理评分数值，也不提供特定分子标志物的定量结果。

Prepare a 1 ml syringe by filling with 15µl 0.5 M EDTA ph 8.0 and then capping with a 23g needle. The syringe will be us Prepare a 1 ml syringe by filling with 15µl 0.5 M EDTA ph 8.0 and then capping with a 23g needle. The syringe will be used for drawing blood from the heart. Prepare a 10 ml syringe with 50-100 µl of heparin (1000 units per ml), then fill the syringe to 10 ml with PBS. Remove a Prepare a 10 ml syringe with 50-100 µl of heparin (1000 units per ml), then fill the syringe to 10 ml with PBS. Remove all the air from the syringe. Cap with a 30g needle. The syringe will be used to flush the heart. Anesthetize the mouse. Anesthetize the mouse. Determine weight and length of the mouse. Determine weight and length of the mouse. Tape the mouse’s arms to 3-4 layers of paper towels and place under a light source. Tape the mouse’s arms to 3-4 layers of paper towels and place under a light source. Cut the skin of the mouse from the abdomen to the top of the thorax. Cut the skin of the mouse from the abdomen to the top of the thorax. Open the abdominal wall below the ribcage. Open the abdominal wall below the ribcage. Lift the sternum with tweezers and cut the diaphragm. Then cut away the lower part of the ribcage to partially expose th Lift the sternum with tweezers and cut the diaphragm. Then cut away the lower part of the ribcage to partially expose the heart. Draw blood from the heart by sticking the needle of the 1 ml syringe (see 1) into the apex of the left ventricle. Draw b Draw blood from the heart by sticking the needle of the 1 ml syringe (see 1) into the apex of the left ventricle. Draw blood by slowly pulling the plunger, twisting the needle. Do not draw for over a minute to avoid blood clots. Transfer the blood into an Eppendorf tube. Mix the tube by inverting it a few times. Centrifuge the tube of blood and transfer the plasma into a fresh tube. Make a small incision in the right atrium for drainage. Make a small incision in the right atrium for drainage. Stick the 10 ml syringe into the apex of the left ventricle and flush the blood from the mouse with 10 ml of PBS. Stick the 10 ml syringe into the apex of the left ventricle and flush the blood from the mouse with 10 ml of PBS. If the liver is to be saved take several sections and immediately freeze at –80ºC. If the liver is to be saved take several sections and immediately freeze at –80ºC. Dry the mouse by turning it over and pressing it against paper towels. Transfer to clean, dry paper towels and re-tape t Dry the mouse by turning it over and pressing it against paper towels. Transfer to clean, dry paper towels and re-tape the arms. Place the mouse under a dissecting microscope. Place the mouse under a dissecting microscope. Remove the remaining ventral part of the ribcage, carefully cut the right clavicle leaving the brachiocephalc artery and Remove the remaining ventral part of the ribcage, carefully cut the right clavicle leaving the brachiocephalc artery and its branches intact. Using microdissection scissors, cut out all of the fat around the ascending aorta and brachiocephalic artery. Cut all the fat and tissue surrounding the heart, including the pulmonary artery, and veins. Cut all the fat and tissue surrounding the heart, including the pulmonary artery, and veins. Flush the heart again through the right ventricle, left atrium, and left ventricle using a 10 ml syringe containing a to Flush the heart again through the right ventricle, left atrium, and left ventricle using a 10 ml syringe containing a total of 3 ml of PBS to clean out residual blood. Cut the brachiocephalic artery distal to where it branches into the right subclavian and right carotid and then cut it a Cut the brachiocephalic artery distal to where it branches into the right subclavian and right carotid and then cut it at its branching site from the aorta so you end up with a Yshaped piece. Put it in a base mold (Fisher cat no 22-038217) and using a 10 ml syringe perfuse it with 1 ml of PBS to get it clean. It is important while handling the brachiocephalic artery to touch it only at its ends and not in the middle. Cut the aorta proximal to the branching site of the brachiocephalic artery. Cut the aorta proximal to the branching site of the brachiocephalic artery. Take out the heart with the aorta. The atria must remain intact as they serve as landmarks when cutting the heart in the Take out the heart with the aorta. The atria must remain intact as they serve as landmarks when cutting the heart in the atherosclerosis assay. Flush the heart-aorta preparation through the aorta with a 10 ml syringe containing 2 ml of PBS and place the heart in a Flush the heart-aorta preparation through the aorta with a 10 ml syringe containing 2 ml of PBS and place the heart in a 20 ml scintillation vial containing 10 ml of formaldehyde solution (10% v/v in aqueous phosphate buffer, Mallinckrodt). Take the cleaned piece of the brachiocephalic artery and place it in a new base mold, fill it up to the first lip of the Take the cleaned piece of the brachiocephalic artery and place it in a new base mold, fill it up to the first lip of the base mold with Tissue Tek O.C.T. compound. Move the brachiocephalic artery around carefully to make sure it fills with OCT. Turn the Y-shaped piece of brachiocephaic artery piece so the prongs of the fork face the bottom of the base mold and fr Turn the Y-shaped piece of brachiocephaic artery piece so the prongs of the fork face the bottom of the base mold and freeze it in that position on a block of dry ice.

**Preparation of Aortic Root and Brachiocephalic Artery for Lesion Quantitation:** **Preparation of Aortic Root and Brachiocephalic Artery for Lesion Quantitation:** 1. Prepare a 1 ml syringe by filling with 15µl 0.5 M EDTA ph 8.0 and then capping with a 23g needle. The syringe will be used for drawing blood from the heart. 2. Prepare a 10 ml syringe with 50-100 µl of heparin (1000 units per ml), then fill the syringe to 10 ml with PBS. Remove all the air from the syringe. Cap with a 30g needle. The syringe will be used to flush the heart. 3. Anesthetize the mouse. 4. Determine weight and length of the mouse. 5. Tape the mouse’s arms to 3-4 layers of paper towels and place under a light source. 6. Cut the skin of the mouse from the abdomen to the top of the thorax. 7. Open the abdominal wall below the ribcage. 8. Lift the sternum with tweezers and cut the diaphragm. Then cut away the lower part of the ribcage to partially expose the heart 9. Draw blood from the heart by sticking the needle of the 1 ml syringe (see 1) into the apex of the left ventricle. Draw blood by slowly pulling the plunger, twisting the needle. Do not draw for over a minute to avoid blood clots. Transfer the blood into an Eppendorf tube. Mix the tube by inverting it a few times. Centrifuge the tube of blood and transfer the plasma into a fresh tube. 10. Make a small incision in the right atrium for drainage. 11. Stick the 10 ml syringe into the apex of the left ventricle and flush the blood from the mouse with 10 ml of PBS. 12. If the liver is to be saved take several sections and immediately freeze at –80°C. 13. Dry the mouse by turning it over and pressing it against paper towels. Transfer to clean, dry paper towels and re-tape the arms. 14. Place the mouse under a dissecting microscope. 15. Remove the remaining ventral part of the ribcage, carefully cut the right clavicle leaving the brachiocephalc artery and its branches intact. Using microdissection scissors, cut out all of the fat around the ascending aorta and brachiocephalic artery. 16. Cut all the fat and tissue surrounding the heart, including the pulmonary artery, and veins. 17. Flush the heart again through the right ventricle, left atrium, and left ventricle using a 10 ml syringe containing a total of 3 ml of PBS to clean out residual blood. 18. Cut the brachiocephalic artery distal to where it branches into the right subclavian and right carotid and then cut it at its branching site from the aorta so you end up with a Y-shaped piece. Put it in a base mold (Fisher cat no 22-038217) and using a 10 ml syringe perfuse it with 1 ml of PBS to get it clean. It is important while handling the brachiocephalic artery to touch it only at its ends and not in the middle. 19. Cut the aorta proximal to the branching site of the brachiocephalic artery. 20. Take out the heart with the aorta. The atria must remain intact as they serve as landmarks when cutting the heart in the atherosclerosis assay. 21. Flush the heart-aorta preparation through the aorta with a 10 ml syringe containing 2 ml of PBS and place the heart in a 20 ml scintillation vial containing 10 ml of formaldehyde solution (10% v/v in aqueous phosphate buffer, Mallinckrodt). 22. Take the cleaned piece of the brachiocephalic artery and place it in a new base mold, fill it up to the first lip of the base mold with Tissue Tek O.C.T. compound. Move the brachiocephalic artery around carefully to make sure it fills with OCT. 23. Turn the Y-shaped piece of brachiocephaic artery piece so the prongs of the fork face the bottom of the base mold and freeze it in that position on a block of dry ice. **Embedding Hearts in Gelatin for Aortic Root Analysis:** **Embedding Hearts in Gelatin for Aortic Root Analysis:** 1. Fix hearts in buffered formalin for 2 weeks at 4°C. 2. Place each heart in an embedding capsule; and place up to 40 capsules in a 1000 ml beaker. Wash under cold running tap water for 1 hour. 3. Make gelatin solutions containing 5%, 10% and 25% gelatin in H²0 and dissolve at 50°C in a water bath. 4. Place washed heart capsules in a shallow plastic container, float container in a 42°C water bath and cover heart capsules with 5% gelatin for 2 hours. 5. Pour back gelatin into a bottle (can be stored at 4°C for future use) and cover hearts with 10% gelatin for 2 hours. 6. Pour back gelatin into a bottle (can be stored at 4°C for future use) and cover hearts with 25% gelatin. Incubate in water bath over night. 7. Place container with heart capsules in refrigerator for 3 hours. 8. Remove heart capsules from solidified gelatin, discard gelatin. 9. Remove embedded hearts from capsules, trim gelatin around the heart to a cube and place trimmed hearts back into buffered formalin. 10. Store at 4°C for a minimum of 24 hours before sectioning the hearts. **Sectioning Hearts for Aortic Root Analysis:** **Sectioning Hearts for Aortic Root Analysis:** 1. Locate the left and right atria. Using a sharp razor-blade cut the bottom half of the heart off in a plane parallel to the atria. **NOTE:** It is essential that the gross cut is parallel to the atria so that a cross section of all three aortic valves is in the same geometric plane. Discard lower half of the heart. 2. Make a second cut above the top of the heart parallel to the first cut on the bottom. 3. Store the heart at 4°C in buffered formalin until ready to section. 4. Mount the trimmed heart on a sample pedestal in the cryostat using OCT compound, the aortic side of the block facing the pedestal. 5. Cover the rest of the gelatin block with OCT and let it freeze. 6. Set section thickness to 30µm and start sectioning until the part of the block that contains the heart tissue is visible. If a complete heart section is not visible, reposition the pedestal. 7. Once a complete heart section is visible, place it on a slide and to check the anatomic location of the section (initially you will see just a thick muscular wall). (**note**: to flatten the section for better viewing under the microscope dip the slide in water). 8. Cut approx. 20 of these 30µm sections and check under the microscope to see if the aortic sinus approaches. The presence of the left and right atria suggests that the sinus is close. The aortic sinus will usually appear rounded, the aortic wall won’t be thick yet and the valves won’t be distinct. Start saving the sections when the valve leaflets become clearly visible. (Reposition the pedestal if necessary to have all 3 leaflets of the valve in one plane) 9. Change thickness of sections to 12 µm and save every other section. Put 4 sections on one slide in this order, and label mouse and slide no: [ms.JPG]]() 10. Cut and save sections until the aorta is rounded and all of the valve leaflets are gone. Usually, this is after 20 to 24 sections (5 to 6 slides). 11. Place slides in a covered glass dish with formalin vapor (put some formalin on bottom of dish) and store slides at 4°C for at least 12 hours prior to staining. **Staining Heart Sections for Aortic Root Analysis:** **Staining Heart Sections for Aortic Root Analysis:** 1. 1 min. H²O 2. 1 min. H²O 3. 30 sec. 60% isopropanol 4. 18 min filtered oil red-O (0.24% in 62.5% 2-propanol) 5. 30 sec. 60% isopropanol rinse 6. 1 min. H²O 7. 1 min. H²O 8. 2 min Harris Hematoxylin 9. 1 min Bluing Solution (H²O with a few drops of ammonia) 10. 1 min. H²O 11. 30 sec. Light Green 12. 1 dip H²O 13. Keep in H²O until ready to put cover slip on the slide 14. Fix cover slips with glycerol gelatin 15. Wash slides to remove excess glycerol gelatin **Sectioning Brachiocephalic Artery for Quantitation of Cross Sectional Lesional Area:** **Sectioning Brachiocephalic Artery for Quantitation of Cross Sectional Lesional Area:** 1. Set cutting temperature of microtome to –30°C. Mount specimens embedded in OCT on cutting pedestal, the opening of the Y shaped vessel should face towards the microtome cutting blade. 2. Set section thickness to 10µm. Start sectioning the right carotid and right subclavian arteries up to the point where they first combine into the brachiocephalic. A section of this point will not have the separation between the two vessels and will be considered the starting point for the following distances. 3. Once this point has been defined, cut 200µm discarding the sections. 4. Use Fisher Colorfrost plus slides to save sections 5. Save the following 2 sections (10µm each) on a the first slide (slide 1) in the following order: [ae.JPG]]() 6. Proceed sectioning and discard the following 80µm (8 sections) 7. Save the next 2 sections (10µm, each) → slide 2 8. Proceed sectioning and discard the following 80µm (8 sections) 9. Save the next 2 sections (10µm, each) → slide 3 10. Proceed sectioning and discard the following 80µm (8 sections) 11. Save the next 2 sections (10µm, each) → slide 4 12. Save 4 additional sections (10µm each), one per slide → slides a,b,c,d The purpose is to have consecutive sections available for immunostaining. These sections are not relevant for quantification and can be discarded if immunostaining will not be done. 13. Proceed sectioning and discard the following 40µm (4 sections) 14. Save the next 2 sections (10µm, each) → slide 5 15. Proceed sectioning and discard the following 80µm (8 sections) 16. Save the next 2 sections (10µm, each) →slide 6 17. Proceed sectioning and discard the following 80µm (8 sections) 18. Save the next 2 sections (10µm, each) →slide 7 [d.JPG]]() **Staining Brachiocephalic Artery Sections for Lesion Quantitation:** **Staining Brachiocephalic Artery Sections for Lesion Quantitation:** a. 1 min. H²O b. 1 min. H²O c. 30 sec. 60% isopropanol d. 18 min filtered oil red-O (0.24% in 62.5% 2-propanol) e. 30 sec. 60% isopropanol rinse f. 1 min. H²O g. 1 min. H²O h. 2 min Harris Hematoxylin i. 1 min Bluing Solution (H²O with a few drops of ammonia) j. 1 min. H²O k. 30 sec. Light Green l. 1 dip H²O m. Keep in H²O until ready to put cover slip on the slide n. Fix cover slips with glycerol gelatin o. Wash slides to remove excess glycerol gelatin **Quantification of Aortic Root and Brachiocephalic Artery Lesion Area by Image Pro Software:** **Quantification of Aortic Root and Brachiocephalic Artery Lesion Area by Image Pro Software:** 1. Use a microscope equipped with a video imaging system attached to a computer with Image Pro software 2. Quantify all 5 slides obtained from one aortic root and seven slides obtained from one brachiocephalic artery, preferentially use section number 1 on each slide. If section number 1 is not readable, go to the second section of the slide. 3. Measure only the area of oil red-O stained lesions on top of the internal elastic lamina, do not quantify any oil red-O stained material underneath the internal elastic lamina. In some cases, lesions may be sheared off the internal elastic lamina and are found in the lumen. Those lesions should also be counted. 4. The atherosclerotic lesion area is expressed as the mean of the lesion areas of one section per slide from the 5 aortic root slides or the 7 brachiocephalic artery slides.

H&E staining **Dewaxing:** Paraffin sections are placed in xylene for 3 x 00:03:00 H&E staining **Rehydration: ** Paraffin sections are placed in - 100% EtOH for 3 x 00:03:00 - 95% EtOH for 1 x 00:03:00 - Rinse with water H&E staining **Hematoxylin staining: ** Paraffin sections are stained with hematoxylin for 00:00:20 and rinse under running water for 00:01:00 H&E staining **Differentiation and bluing:** Paraffin sections are placed into the bluing reagent for 00:01:00 and rinse under running water for 00:01:00 _This step results in a blue coloration of the nuclei._ H&E staining **Eosin staining:** Parrafin sections are counterstained with Eosin for 00:00:10 _This step results in a pink coloration of the cytoplasmic components and connective tissue fibers._ H&E staining **Dehydration and Clearing:** Paraffin sections are placed in - 95% EtOH for 00:02:00 - 100% EtOH for 2 x 00:02:00 - Xylene for 2 x 00:02:00 H&E staining **Mounting:** Parrafin sections are mounted with a glass coverslip using Permount.

禁食 Fasting begins first thing in the morning (around 9am). Fast mice for 4-6 hours before OGTT begins. Transfer mice to clean cage and wire top. Keep water bottles during the fasting period. 1. If using High Fat diet (HFD), save food to give back at the end of the OGTT. 1. mark tails with a sharpie for easier identification during OGTT. 口服葡萄糖负荷 Weigh mouse to obtain body weight for dose calculations. Calculate bolus dose of glucose (**1g/kg**). Weight of mouse (g) x 2 = ul sterile glucose (50% dextrose) solution *If using HFD for 4 weeks or more, use 0.5g/kg (weight of mouse (g) x 1 = ul sterile glucose (50% dextrose) solution) 口服葡萄糖负荷 Prepare syringes using 1cc syringe and 18G oral gavage needle. Load all syringes with calculated glucose dose and position in front of home cage. 口服葡萄糖负荷 Prior to the delivery of the sterile glucose (50% dextrose) solution, a time “zero” blood glucose level must be measured. Restrain mouse and clean tail with 70% ethanol/gauze. Extract a small amount of blood from the tail vein onto One Touch Ultra blood glucose strips used with the One Touch Ultra 2 blood glucose monitoring system. Blood can be collected into microvette tubes that can be used to extract plasma which can be assayed for plasma insulin (see step 9 for plasma collection). Microvette tubes are blood collection tubes with a capillary. Tap tube until all the blood is collected in the tube. Place on ice until plasma collection. 口服葡萄糖负荷 Administer the correct dose of sterile glucose (50% dextrose) solution using the syringe and gavage needle. - Restraint of the Mouse, avoiding an excessively tight grip of the mouse, as this will induce a panic response. Ensure that the mouse cannot bite you and cannot move its head. - Gravity alone should be used to move the cannula down the esophagus. While gliding the cannula, rotate slightly as the cannula goes past the epiglottis and into the esophagus.The mouse should swallow the cannula, and this can be observed. - Inject the fluid slowly. - The cannula is then gently removed and the animal is returned to its cage. 口服葡萄糖负荷 Begin timing from the point of successfully delivering the sterile glucose (50% dextrose) solution. 口服葡萄糖负荷 Test and record the blood glucose level (see step 4) at 15, 30, 60, 90 and 120 minutes after the initial glucose delivery. Record each value and the condition of the animal on the Glucose Tolerance Test record sheet. Extract a small amount of blood from the tail vein at 15, 30, 60 and 120 minutes for plasma; skip blood collection at 90 minutes to ensure not to exceed 0.007 ml blood/ g body weight. 口服葡萄糖负荷 Return food and environmental enrichments to cages. 血浆采集 Place tubes into pre-chilled centrifuge. Centrifguge for 2 mins at 2000 rpm. 00:02:00 4 °C 血浆采集 Remove the callipary-lid and dispose in biohazard bin. Place the base lid and seal the microvette (click position). 血浆采集 Centrifuge at 4°C for 10 mins at 10 000 rpm. 00:10:00 4 °C 血浆采集 Using a pipette, transfer the plasma (top transparent layer) to a clean and labelled tube. Store plasma samples at -20°C until assayed. 血浆胰岛素测定 Assay plasma insulin with the STELLUX Chemi Rodent Insulin ELISA kit (ALPCO #80-INSMR-CH01) according to the manufacturer's protocol. Include the kit standards and quality controls in each assay and read the plate by the kit's chemiluminescence procedure.

脱蜡（Dewaxing） 石蜡切片置于二甲苯中脱蜡 3 次，每次 00:03:00。 复水（Rehydration） 石蜡切片依次置于 100% 乙醇中 3 次、每次 00:03:00，再置于 95% 乙醇中 1 次、00:03:00，随后用水漂洗。 苏木素核染色（Hematoxylin staining） 石蜡切片以苏木素染液染色 00:00:20，随后在流水下冲洗 00:01:00。 分化与返蓝（Differentiation and bluing） 石蜡切片置于返蓝液中 00:01:00，随后在流水下冲洗 00:01:00；此步使细胞核呈现蓝色。 伊红染色（Eosin staining） 石蜡切片以伊红复染 00:00:10；此步使细胞质成分与结缔组织纤维呈现粉红色。 脱水与透明（Dehydration and Clearing） 石蜡切片依次置于 95% 乙醇中 00:02:00、100% 乙醇中 2 次、每次 00:02:00，随后置于二甲苯中 2 次、每次 00:02:00。 封片（Mounting） 石蜡切片以 Permount 封片介质加盖玻片封固。