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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, 10461 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.

材料与试剂

 

1.         对照: 已知的阳性对照组织 (如脾脏).

2.         福尔马林

3.         乙醇

4.         亚铁氰化钾

5.         盐酸

6.         核固红(Kernechtrot)

7.         硫酸铝

 

设备

 

1.         微波炉

2.         无酸玻璃管

3.         非金属钳子

程序

 

安全提示: 戴手套,护目镜和穿实验服  避免接触和吸入亚铁氰化钾;只要没有加热它是低毒性的,否则它将释放氰化物气体。

盐酸; 作用与生殖系统和胎儿组织,对皮肤眼睛和呼吸系统有刺激性

1.         去除组织切片的石蜡并且用蒸馏水水化  (使用2 Histoclear 试剂, 100% 乙醇 2, 95%乙醇 1, 90% 乙醇 1, 80%乙醇 1, 70% 乙醇 1, dH2O 2. 每个溶液中放置3-5分钟)

2.         立即转移切片于工作液中(避免组织干燥)或是使用微波法(30秒,通风厨中片置于溶液中5分钟)或是使用传统的方法(室温30分钟)进行处理。

3.         在蒸馏水中洗涤。

4.         核固红染色 (室温孵育5分钟)

5.         水洗若干次。

6.         脱水, 清除(逆转第一步的顺序)

7.         加入固定液和盖玻片。

8.         结果:

铁离子 (血黄铁质):   蓝色

:                       红色

背景:                  粉红色

(切片显示400×目镜下的鼠脾脏)

 

 

配制试剂

 

1.         固定液: 10% 福尔马林

2.         5% 亚铁氰化钾:

亚铁氰化钾 25.0 mg

蒸馏水 500.0 ml

混匀, 倾倒入无酸的棕色瓶中。

6个月可稳定保存。

注意: 不被加热时是低毒性的

3.         5%盐酸:

无水盐酸25.0 ml

蒸馏水475.0 ml

混匀, 倾倒入棕色瓶中。

6个月稳定保存。

注意: 具腐蚀性,避免接触和吸入。

4.         核固红染液 (Kernechtrot)

硫酸铝 25.0 gm

蒸馏水 500.0 ml

核固红 0.5 mg (0.5 gm for 克数 (不是毫克

蒸馏水中溶解硫酸铝,然后加入核固红,加热溶解,待完全溶解冷却过滤,加入微量的麝香草用于保存

标明日期。可稳定保存1年。

注意: 具刺激性,避免接触和吸入。

5.         工作液:

5% 亚铁氰化钾25.0 ml

5% 盐酸25.0 ml

现配现用, 使用后丢弃。

注意: 避免接触和吸入。

 

参考文献

 

1.         Sheehan D., Hrapchak B. (1980). Theory and practice of histotechnology. 2nd ed.; Battelle Press: Ohio, p 217-18.

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.

 

 

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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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