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Purpose: To demonstrate ferric iron in tissue sections. Small amounts of iron are found normally in spleen and bone marrow. Excessive amounts are present in hemochromatosis, with deposits found in the liver and pancreas, hemosiderosis, with deposits in the liver, spleen, and lymph nodes.
Principle: The reaction occurs with the treatment of sections in acid solutions of ferrocyanides. Any ferric ion (+3) in the tissue combines with the ferrocyanide and results in the formation of a bright blue pigment called 'Prussian blue" or ferric ferrocyanide.

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Iron - Prussian Blue Reaction - Mallory’s Method
铁-普鲁士兰反应法- Mallory’s 方法

细胞生物学 > 组织分析 > 组织染色
作者: Hani Jouihan
Hani JouihanAffiliation: Division of Endocrinology, Gerontology and Metabolism, Stanford University School of Medicine, Stanford, USA
For correspondence: hanijouihan@hotmail.com
Bio-protocol author page: a52
Vol 2, Iss 13, 7/5/2012, 11543 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.222

[Abstract] Purpose: To demonstrate ferric iron in tissue sections. Small amounts of iron are found normally in spleen and bone marrow. Excessive amounts are present in hemochromatosis, with deposits found in the liver and pancreas, hemosiderosis, with deposits in the liver, spleen, and lymph nodes.
Principle: The reaction occurs with the treatment of sections in acid solutions of ferrocyanides. Any ferric ion (+3) in the tissue combines with the ferrocyanide and results in the formation of a bright blue pigment called 'Prussian blue" or ferric ferrocyanide.

[Abstract] 目的: 为了显示组织剖面的铁离子。在脾脏和骨髓中存在少量的铁离子,但是在血色沉着病中呈现出大量的铁离子,同时在肝脏和胰腺中发现了沉积,血色沉着病是表现为肝脏,脾脏和淋巴结的三价铁离子的沉积。
原理: 反应发生在酸性亚铁氰化物溶液处理过的剖面组织中。组织中的任何的(+3)铁离子能够与亚铁氰化物结合并且生成亮蓝色的颜色,叫做亚铁氰化铁的“普鲁士兰” 。

Materials and Reagents

  1. Control: A known positive control tissue (such as spleen)
  2. Formalin
  3. EtOH
  4. Hydrochloric acid
  5. Aluminum sulfate
  6. Histoclear reagent
  7. Mounting solution
  8. Fixative (see Recipes)
  9. 5 % potassium Ferrocyanide (see Recipes)
  10. 5% hydrochloric acid (see Recipes)
  11. Nuclear-fast red (Kernechtrot) (see Recipes)
  12. Working solution (see Recipes)

Equipment

  1. Microwave oven
  2. Acid-cleaned glassware
  3. Non-metallic forceps.
  4. Gloves, goggles and lab coat
  5. Fume hood

Procedure

Safety: Wear gloves, goggles and lab coat. Avoid contact and inhalation. Potassium ferrocyanide; Low toxicity as long as it is not heated, it will release cyanide gas.
Hydrochloric acid; target organ effects on reproductive system and fetal tissue. Irritant to skin eyes and respiratory system.

  1. Deparaffinize tissue slide and hydrate to distilled water (use Histoclear reagent 2x, 100% EtOH 2x, 95% EtOH 1x, 90% EtOH 1x, 80% EtOH 1x, 70% EtOH 1x, ddH2O 1x. leave 3-5 min in each solution).
  2. Immediately transfer slides into working solution (avoid drying the tissue) and either use the microwave method (30 sec, then allow slides to stand in solution for 5 min in fume hood) or use the conventional method (room temperature for 30 min).
  3. Rinse in distilled water.
  4. Nuclear-fast red (incubate for 5 min at room temperature).
  5. Wash in tap water (several washes).
  6. Dehydrate, clear (reverse the order of step 1 above).
  7. Add mounting solution and coverslip.
  8. Results:
    Iron (hemosiderin):
    Blue
    Nuclei:
    Red
    Background:
    Pink
    (Slide shows mouse spleen at 400x)

Representative Data



Figure 1. Typical results from this experiment

Recipes

  1. Fixative
    10% formalin
  2. 5% potassium ferrocyanide
    Potassium ferrocyanide 25.0 mg
    Distilled water 500 ml
    Mix well, pour into an acid-cleaned brown bottle.
    Stable for 6 months.
    Caution: Low toxicity if not heated.
  3. 5% hydrochloric acid
    Hydrochloric acid, conc. 25.0 ml
    Distilled water 475.0 ml
    Mix well, pour into brown bottle.
    Stable for 6 months.
    Caution: Corrosive, avoid contact and inhalation.
  4. Nuclear-fast red (Kernechtrot)
    Aluminum sulfate 25.0 mg
    Distilled water 500 ml
    Nuclear-fast red 0.5 mg
    Dissolve the aluminum sulfate in distilled water, then the nuclearfast red, using heat. Cool, filter, and add a few grains of thymol as a preservative.
    Label with date. Stable for 1 year.
    Caution: Irritant, avoid contact and inhalation.
  5. Working solution
    5% potassium ferrocyanide 25.0 ml
    5% hydrochloric acid 25.0 ml
    Make fresh, discard after use.
    Caution: Avoid contact and inhalation.

Acknowledgments

This protocol was adapted/modified from original versions as described in Luna (1968).

References

  1. Sheehan, D., Hrapchak, B. (1980). Theory and practice of histotechnology. 2nd ed.; Battelle Press: Ohio, p. 217-218.
  2. Luna, L. (1968). Manual of histologic staining methods of the AFIP. 3rd ed.; McGraw-Hill: NY. P. 183.
  3. Crookham, J., Dapson, R. (1991). Hazardous chemicals in the histopathology laboratory. 2nd ed.; Anatech.

材料和试剂

  1. 控制:已知阳性对照组织(如脾)
  2. 福尔马林
  3. EtOH
  4. 盐酸
  5. 硫酸铝
  6. 组织清蛋白试剂
  7. 安装解决方案
  8. 固定剂(见配方)
  9. 5%的亚铁氰化钾(见配方)
  10. 5%盐酸(见配方)
  11. 核快红(Kernechtrot)(参见配方)
  12. 工作解决方案(参见配方)

设备

  1. 微波炉
  2. 酸洗玻璃器皿
  3. 非金属钳。
  4. 手套,护目镜和实验室外套
  5. 通风橱

程序

安全:戴上手套,护目镜和实验室外套。 避免接触和吸入。 亚铁氰化钾; 低毒性只要不加热,就会释放氰化物气体 盐酸; 靶器官对生殖系统和胎儿组织的影响。 刺激皮肤眼睛和呼吸系统。

  1. 将组织载玻片和水合物脱石蜡至蒸馏水(使用Histoclear试剂2x,100%EtOH 2x,95%EtOH 1x,90%EtOH 1x,80%EtOH 1x,70%EtOH 1x,ddH 2 O 1x 。在每种溶液中离开3-5分钟)。
  2. 立即将玻片转移到工作溶液中(避免干燥组织),并使用微波法(30秒,然后让载玻片在通风橱中在溶液中静置5分钟)或使用常规方法(室温30分钟) br />
  3. 用蒸馏水冲洗。
  4. 核快速红色(在室温下孵育5分钟)
  5. 在自来水(几次洗涤)中洗涤。
  6. 脱水,清除(与上述步骤1的顺序相反)。
  7. 添加安装溶液和盖玻片。
  8. 结果:
    铁(含铁血黄素):
    蓝色
    核:
    红色
    背景:
    粉红色
    (幻灯片以400x显示小鼠脾脏)

代表数据



图1.此实验的典型结果

食谱

  1. 固定剂
    10%福尔马林
  2. 5%的亚铁氰化钾 亚铁氰化钾25.0mg
    蒸馏水500 ml
    混合均匀,倒入酸性清洗的棕色瓶中。
    稳定6个月。
    注意:如果不加热,毒性低。
  3. 5%盐酸
    盐酸, 25.0 ml
    蒸馏水475.0 ml
    混合均匀,倒入棕色瓶中 稳定6个月。
    注意:腐蚀,避免接触和吸入
  4. 核快红(Kernechtrot)
    硫酸铝25.0mg
    蒸馏水500 ml
    核坚牢红0.5mg
    将硫酸铝溶解在蒸馏水中,然后用核快速红色,加热。 冷却,过滤,并加入几粒百里酚作为防腐剂。
    标签与日期。 稳定1年。
    注意:刺激,避免接触和吸入
  5. 工作解决方案
    5%氰亚铁酸钾25.0ml
    5%盐酸25.0ml
    新鲜,使用后丢弃。
    注意:避免接触和吸入。

致谢

该协议从Luna(1968)中描述的原始版本改编/修改。

参考文献

  1. Sheehan,D.,Hrapchak,B。(1980)。 组织技术的理论与实践。 第2版; Battelle Press:Ohio,p。
  2. Luna,L。(1968)。 AFIP的组织学染色方法手册。 第3版; McGraw-Hill:NY。
  3. Crookham,J.,Dapson,R。(1991)。 危险化学品在组织病理学实验室。 第2版; Anatech。
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How to cite this protocol: Jouihan, H. (2012). Iron - Prussian Blue Reaction - Mallory’s Method. Bio-protocol 2(13): e222. DOI: 10.21769/BioProtoc.222; Full Text



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