天狼星红（Picrosirius red）胶原染色

Scope and method

Sample：formalin-fixed paraffin-embedded tissue sections

染色

• db/db mouse on methionine-choline-deficient diet

  Age

  as reported in cited studies

  Tissue

  liver

• ALIOS (American lifestyle-induced obesity syndrome) model: Western diet and sedentary behaviour-induced NASH mouse

  Age

  as reported in cited studies

  Tissue

  liver

• DIAMOND model: Western diet with glucose-fructose sugar water-induced NASH in B6/129 mouse

  Age

  as reported in cited studies

  Tissue

  liver

• Atherogenic diet-fed C57BL/6J mouse

  Age

  as reported in cited studies

  Tissue

  liver

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

  Age

  as reported in cited studies

  Tissue

  liver

• Western diet-induced NASH C57BL/6J mouse

  Age

  as reported in cited studies

  Tissue

  liver

• methionine-choline-deficient (MCD) diet-fed mouse

  Age

  as reported in cited studies

  Tissue

  liver

• choline-deficient L-amino acid-defined (CDAA) diet-fed mouse

  Tissue

  liver sections

• 高脂高果糖饮食诱导代谢综合征模型（C57BL/6 小鼠）

  Age

  来源研究取材时点

  Tissue

  kidney

• CC G×E 多品系队列 (NZO 背景)

  Tissue

  liver

• CC042 重度 MASH 模型

  Tissue

  liver

• CDAHFD 饮食诱导 NASH 模型（C57BL/6J 小鼠）

  Age

  6 周饮食

  Sex

  Male

  Tissue

  liver sections

Reagents and materials

• 自来水（组织学染色用）
• 1% 盐酸乙醇分化液（acid alcohol）
• 自来水（组织学染色用）
• Weigert 苏木素染色液（A/B 现配）
• 蒸馏水（aqua dest，组织学染色用）
• 蒸馏水（aqua dest，组织学染色用）
• 蒸馏水（aqua dest，组织学染色用）
• 0.2% 磷钨酸溶液（PTA）
• 组织学二甲苯（混合异构体）
• 蒸馏水（aqua dest，组织学染色用）
• 95% 乙醇溶液
• 70%乙醇
• Ethanol
• Permount 组织学封片剂（Fisher SP15）
• 组织学二甲苯（混合异构体）
• 组织学二甲苯（混合异构体）
• Ethanol
• Ethanol
• Ethanol
• 0.5% 乙酸分化液
• 天狼星红染液

Procedure

1. 脱蜡与水化

   Dewax and rehydrate tissue sections

2. 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.

3. 磷钨酸预处理

   Rinse with distilled water

4. 磷钨酸预处理

   Rinse with distilled water

5. 磷钨酸预处理

   Rinse with distilled water

6. 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

7. 核染色

   Wash with running tap water to remove excess stain

8. 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

9. 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.

10. 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.

11. 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

12. 天狼星红分化

    Drain acetic acid solution from the slides as much as possible

13. 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

14. 脱水、透明与封片

    100% ethanol Ethanol POCD Scientific Catalog #ETHABS20M

15. 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

16. 脱水、透明与封片

    100% Xylene Xylene POCD Scientific Catalog #XYL5P

17. 脱水、透明与封片

    100% Xylene Xylene POCD Scientific Catalog #XYL5P

18. 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

19. 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]](

Quality control and readouts

切片脱蜡水化完全；磷钨酸预处理后充分冲洗；Weigert 苏木素现配现用，分化后充分水洗以避免铁离子带入天狼星红液；天狼星红/苦味酸溶液保持湿润并避免金属容器；用显微镜检查胶原呈红色、细胞呈黄色，偏振光下胶原呈双折射。

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。

Study design

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。

Technical services