STAM (neonatal streptozotocin injection followed by high-fat diet) mouse

石蜡包埋组织切片的天狼星红胶原染色。按源方案完成脱蜡水化、可选磷钨酸预处理、可选 Weigert 苏木素核染色、天狼星红/饱和苦味酸染色、乙酸分化、脱水透明与树脂封片；染色时间和分化程度按具体组织优化。样本来源、分组与切片数量按各研究设计确定。

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。

明场下胶原纤维呈红色，骨呈红色，肌肉、上皮和红细胞呈黄色，细胞核呈棕黑色；偏振光下胶原与骨呈双折射。结果用于胶原的定性或半定量观察；本方案不包含完整的纤维化面积、分期或 I/III 型胶原亚型定量。

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。

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。

脱蜡与水化 Dewax and rehydrate tissue sections 磷钨酸预处理 Incubate in 0.2% phosphotungstic acid (PTA) solution PTA solution: 0.2 g PTA in 100 ml distilled water Phosphotungstic acid hydrate Merck MilliporeSigma (Sigma-Aldrich) Catalog #P4006 This PTA pre-treatment prevents non-specific nuclear staining in PSR, thereby enhancing the distinction between collagen and cellular staining. Elimination of the non-specific staining is particularly beneficial if performing automated digital image analysis, improving rigour of the method. 磷钨酸预处理 Rinse with distilled water 磷钨酸预处理 Rinse with distilled water 磷钨酸预处理 Rinse with distilled water 核染色 Stain in Weigert’s Haematoxylin (combine solution A & B immediately before use) Solution A: 1 g Haematoxylin in 100 ml of 95% Ethanol Solution B: 1.6 g Ferric chloride hexahydrate (Iron III chloride; FeCl3.6H2O) + 95 ml distilled water + 1 ml concentrated HCl Haematoxylin Fronine Laboratory Supplies Catalog #JL555S Ethanol Hurst Scientific Catalog #EIMS95-5L Iron (III) chloride hexahydrate Merck MilliporeSigma (Sigma-Aldrich) Catalog #F2877 Hydrochloric Acid Banksia Scientific Company (AJAX FineChem) Catalog #AJA256 核染色 Wash with running tap water to remove excess stain 核染色 Differentiate with acid alcohol (5-10 dips) to clear the slides [1% Hydrochloric Acid (HCl) in 70% Ethanol] Hydrochloric Acid Banksia Scientific Company (AJAX FineChem) Catalog #AJA256 Ethanol POCD Scientific Catalog #ETHABS70WV5 核染色 Wash with running tap water It is important to ensure that the slide is completely cleared of excess Weigert's Haematoxylin at this point. If there is carryover into the PSR solution, it becomes contaminated with the ferric chloride, resulting in non-specific red background staining. 胶原染色 Stain with picrosirius red solution Solution: 0.1 g Sirius Red F3BA + 100 ml saturated aqueous picric acid 1.3 Mass Percent, in distilled water Sirius red F3BA BDH Chemicals Ltd Poole England Catalog #34149 CI: 35780 Picric acid May & Baker LTD Sirius red is also known as "Direct Red 80" Solution can be re-used many times. Lasts 20 years. Picric acid is an unstable explosive when dry, where it becomes highly sensitive to shock, heat and friction. Ensure it remains hydrated at all times. Picric acid reacts with metals to form unstable picrate salts which are more explosive. Do not use metal containers, lids or racks. Picric acid is toxic if swallowed, inhaled or absorbed through the skin. Consult the MSDS and follow all recommended safety precautions. 天狼星红分化 Differentiate the PSR staining with 2 changes of a 0.5% acetic acid solution, 5-10 dips each. The timing of this differentiation (or number of dips) should be optimised for each specific tissue. Use a microscope to check that collagen is stained red and cells are stained yellow. Solution: 0.5 ml glacial acetic acid + 99.5 ml distilled water Glacial acetic acid Ajax Finechem (ThermoFisher Scientific) Catalog #AJA1-500ML 天狼星红分化 Drain acetic acid solution from the slides as much as possible 脱水、透明与封片 100% ethanol Note: If you want to maintain the yellow cytoplasmic staining, ensure the dehydration is quick. 10 dips per ethanol step is usually sufficient. Ethanol POCD Scientific Catalog #ETHABS20M 脱水、透明与封片 100% ethanol Ethanol POCD Scientific Catalog #ETHABS20M 脱水、透明与封片 100% ethanol Note: ensure whole slide and slide rack is fully immersed in ethanol to remove all traces of water, preventing water contamination of the xylene Ethanol POCD Scientific Catalog #ETHABS20M 脱水、透明与封片 100% Xylene Xylene POCD Scientific Catalog #XYL5P 脱水、透明与封片 100% Xylene Xylene POCD Scientific Catalog #XYL5P 脱水、透明与封片 Coverslip with a resinous mounting medium, such as DPX DPX Mountant for histology Merck MilliporeSigma (Sigma-Aldrich) Catalog #06522 结果 Collagen: Red Bone: Red Muscle: Yellow Epithelium: Yellow Erythrocytes: Yellow Nuclei: Brown - Black Note - collagen and bone stained specifically with Sirius Red are birefringent, enabling identification and quantification under polarised light microscopy. [GAA_no W 10x 2.jpeg]]( [GAA_no W 10x 2_Pol.jpeg]]( [GAA_W 20x.jpeg]]( [GAA_W 20x_Pol.jpeg]](

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。