db/db (Lepr^db)

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。

小鼠腹腔注射胰岛素后的血糖反应检测。禁食 4–6 小时；按 1–1.2 U/kg 给予胰岛素（源方案以 100 U/mL 原液经冷 PBS 两步稀释，注射体积按体重计算）；注射前及注射后 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。

以预先生成的 cDNA 为输入的 SYBR Green 实时定量 PCR 检测。每孔加入 10 ng cDNA，技术三重复；使用 PowerUp SYBR Green Master Mix 与 5 µM 正反向引物；按源方案循环条件扩增并读取 CT，以看家基因和参考实验组进行 ΔΔCT 分析。具体基因、引物和生物学分组按各研究设计确定。

FFPE 肾组织切片的 PAS 染色流程，按源方案 FFPE 分支执行脱蜡、复水、氧化、Schiff 显色、苏木素复染、脱水透明和封片。具体切片厚度、视野与定量方法按研究设计确定。

以血糖浓度对时间作图得到完整血糖-时间曲线，并可计算曲线下面积（AUC）反映葡萄糖清除程度；当各受试对象基线血糖不同时应比较高于基线的幅度或曲线下面积，而非仅比较单一终点值。该检测反映葡萄糖负荷后的整体清除，不等同于胰岛素作用的直接测量。

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。

基线与注射后 15、30、60、90、120 分钟血糖时间序列；可计算血糖下降幅度或曲线下面积。结果反映外源胰岛素负荷后的整体血糖反应，不单独等同于组织特异性胰岛素敏感性。

各样品目标序列的 CT 值；以看家基因归一化并以参考实验组计算 ΔΔCT/相对表达量。本方案不包含具体基因或疾病特异结论，输出判读依赖所选的引物、看家基因和实验分组。

340 nm吸光度变化对应的葡萄糖浓度。

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。

肾小球基底膜、系膜基质和糖蛋白呈 PAS 阳性（品红/紫红色），细胞核呈蓝色；按研究设计进行肾小球系膜扩张或病变范围判读。

输出为每样品尿白蛋白浓度（mg/l），按标准曲线插值报告；该读数本身不是白蛋白/肌酐比值，换算比值需另测尿肌酐。

提前一周单笼饲养 实验前一周将小鼠改为单笼饲养。 称量体重 逐只称量小鼠体重，用于按体重计算给糖体积。 禁食6小时 禁食六小时（06:00:00）。 测定基线血糖 禁食结束后，先用血糖仪测定血糖作为基线。 腹腔注射葡萄糖 按2 g/kg体重腹腔注射葡萄糖：将2 g D-葡萄糖溶于10 ml生理盐水配成20%葡萄糖贮备液，按每克体重10 ul给予。 系列采血测定血糖 于注射葡萄糖后15、30、45、60、90和120分钟采集血样，用血糖仪测定血糖。

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.

禁食 Fasting begins first thing in the morning (around 9am). Fast mice for 4-6 hours before ITT 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 ITT. 1. Mark tails with a sharpie for easier identification during ITT. 胰岛素溶液配制 Stock 1: 50ul of 100U/ml Humulin R into 4950ul of cold sterile 1X PBS. Invert solution 3-4 times to mix. Stock 2: 1ml of Stock 1 into 9ml of cold sterile 1X PBS. Invert solution 3-4 times to mix. Keep on ice until use 胰岛素负荷 Weigh mouse to obtain body weight for dose calculations. Calculate bolus dose of insulin (**1-1.2 units/kg**). Weight of mouse (g) x 10 = ul insulin stock 2 solution 胰岛素负荷 Prepare syringes using 1cc syringe and 26G needle. Load all syringes with calculated insulin dose and position in front of home cage. 胰岛素负荷 Prior to the delivery of the insulin 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. 胰岛素负荷 Administer the correct dose of Insulin solution using an IP (intraperitoneal) injection method. - Restrain the mouse. Then place the mouse is in dorsal recumbency with the head down allowing the viscera to move cranially. - Using the bend of the knee and the ventral abdominal midline as landmarks, the needle is inserted half way between the midline and lateral side of the animal at the natural bend of the knee. Insert the needle at a 30 to 45 degree angle into either the left or right lower abdominal quadrant. - Prior to injection, aspirate to make sure the needle has not penetrated a blood vessel, the intestines or the urinary bladder. When aspirating the syringe, you should see an air bubble in the hub of the needle, and not any form of fluid. - Inject and return mouse to cage. 胰岛素负荷 Begin timing from the point of successfully delivering the insulin solution. 胰岛素负荷 Test and record the blood glucose level at 15, 30, 60, 90, and 120 minutes after the insulin injection. Record each value and the condition of the animal on the Insulin Tolerance Test record sheet. 胰岛素负荷 Return food and environmental enrichments to cages.

qPCR 反应体系 **Option 2****: SYBR qPCR** qPCR 反应体系 Prepare SYBR Master mix (ul per well): 1. 5ul PowerUp SYBR Green Master Mix (#A25776, Applied Biosystem-ThermoFisher) 1. 1ul Primers (Forward and Reverse, diluted in Nuclease-free water at 5µM) qPCR 反应体系 Add 6ul of SYBR Master mix to each well with an automatic pipette (Multipette E3, Eppendorf) qPCR 反应体系 Add 4ul of cDNA per well (10ng cDNA) in technical triplicates to each well of LightCycler 480 Multiwell 384-plate (#4729749001, Roche Diagnostics) 热循环 Perform PCR using the following cycling conditions in a LightCycler® 480 System (Roche): 1. 50˚C for 2min 1. 95˚C for 2min 1. 95˚C for 15s + 60˚C for 1min (x40cycles) 数据分析 Using the LightCycler Software obtain the threshold cycles (CT) signals from each samples. 数据分析 Perform ΔΔCT-method to analyze the expression data using the endogenous control genes and the reference experimental group.

Calibrate Cobas for Glucose analysis by running a multi analyte standard and two control serum. Calibrate Cobas for Glucose analysis by running a multi analyte standard and two control serum. Sample Handling as performed by the Cobas Mira Plus. Sample Handling as performed by the Cobas Mira Plus. a) Pipette 3 µL of sample into cuvette. b) Absorbance is measured at 340 nm. c) Add 100 µL of Glucose liquid reagent. d) Mixture is incubated at 37ºC for 10 minutes. e) Absorbance is measured at 340 nm. Change in absorbance is calculated.

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。

Start with FFPE here: Allow PAS “kit” to come to room temperature on the bench. Start with FFPE here: For paraffin sections, deparaffinize in xylene, two changes, 00:03:00 each. Start with FFPE here: Hydrate through graded alcohols, 00:01:00 each: 100%, 100%, 95%, 70%, water Start with FFPE here: Rinse well in distilled water by holding finger over slides, pouring water into sink and adding water. Do this 5 times. Start Frozen samples here: Place in 0.5% Periodic acid for 00:10:00 (to oxidize). Start Frozen samples here: Rinse well in distilled water (follow Step 4). Start Frozen samples here: Pour Schiff’s reagent into Coplin jar containing slides. Allow to sit 15-30 minutes at room temperature (kidney samples ~30 minutes). Start Frozen samples here: Rinse in running warm (~40 °C) distilled or tap water (follow Step 4). The tissue should appear pink after rinsing with warm water. Start Frozen samples here: Place slides in movable gray slide holder. Start Frozen samples here: Counterstain in Hematoxylin (staining line) for 00:01:00. Start Frozen samples here: Rinse well in distilled water, starting with blue container next to hematoxylin (follow Step 4). Start Frozen samples here: Quickly dip slides into “Bluing” agent (0.5% ammonium hydroxide). Start Frozen samples here: Rinse 1 minute in distilled water until pink color is visible (follow Step 4). Start Frozen samples here: Dehydrate through graded alcohols, 10 short dips in each: 95%, 95%, 100%, 100% If the last 100% ethanol rinse is colored by residual stain, additional washing in new 100% ethanol solution should be performed. Start Frozen samples here: Fix in 2 rounds of xylenes, 00:01:00 each (in the hood). Start Frozen samples here: Coverslip slides: - Place coverslip on paper towel - Add 2-3 drops of cytoseal to edge (depending on size) - Dip slide into xylenes, take out and roll the xylene lengthwise on slide - Line up slide and cover slip and slowly place the slide on the coverslip - If needed, use dissecting tool to remove bubbles Start Frozen samples here: Image slides using a Leica brightfield scanner, AxioScan Z1 slide scanner (or equivalent) at 20x (~1 µm spatial resolution) and save as.tiff or.jpg file.

Prepare working reagent. Prepare working reagent. Prepare standards by serially diluting 200 mg/l standard 1:1 to make 100, 50, 25, 12.5, 6.25 standards. Prepare standards by serially diluting 200 mg/l standard 1:1 to make 100, 50, 25, 12.5, 6.25 standards. Add 25 μl of standard and sample to each well. Add 25 μl of standard and sample to each well. Add 125 μl of working reagent. Read in fluorimeter using 590 nm excitation and 616 nm emission. Add 125 μl of working reagent. Read in fluorimeter using 590 nm excitation and 616 nm emission. _**IMPORTANT: **__Make sure not to add any bubbles to the wells when dispensing reagents, this will interfere with reading in the platereader._ Use a polynomial 2nd order curve fit to construct a standard curve. Interpolate the values of the unknowns using the sta Use a polynomial 2nd order curve fit to construct a standard curve. Interpolate the values of the unknowns using the standard curve.