Special Stains

Subject: Histopathology

Hi guys!
This is my 7th week at Histopathology, and so we have gotten pretty familiar at how things are managed and operated there. My previous post mentioned a bit on Special Stains, so now I'm here to share about 2 of the more common ones.

*Note:

• Commerical reagents are usually used in the lab
  
• Timings in the staining procedure are for your reference only because I've learnt that different types and different sections of tissues absorb and expel dye molecules at different rate.
  So if you are staining 5 slides at the same time using the same timing, the results obtained for each slide will vary. Thus, it is important to perform microscopic examination for each slide after each staining.
  However, there are also some stains which can be left on the sections as long as you want as the duration does not affect the staining.
  
• All reagents have their specific "lifespan" so it is important to record the date when the bottle of reagent is opened and their expiry date. Reagents are changed as often as needed to ensure quality staining.
  
• All types of staining including H&E are to be done with a control (positive control) section. It can either be at the upper half of the same slide, or on another new slide. This is added for the pathologist as a reference during microscopic examination.

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Alcian Blue (ALBL)

Function:
· Stains acid mucins and acid mucosubstances

Where can acid mucins and acidic mucosubstances be found?
· Excessive amount of non-sulphated acidic mucosubstances can be seen in mesothelioma, with certain
amount occurring normally in blood vessel walls but increased amount in early lesion of atherosclerosis.

Principle:
· Alcian Blue is a group of polyvalent basic dyes that are water soluble
· Blue colouration is present due to presence of copper in the dye molecule
· Alcian Blue at pH 2.5 stains both sulphated and carboxylated sialomycins (gylcoproteins)
· Alcian Blue forms self-linkages with acidic groups of acid mucopolysaccharides

Control used:
· Small intestine or colon

Fixative used:
· 10% Neutral Buffered Formalin

Chemicals required:
· 1% Alcian Blue
· 3% aqueous Glacial Acetic Acid
· Nuclear Fast Red

Reagent preparation:

1. Alcian Blue
· Alcian blue – 1g
· 3% aqueous Glacial Acetic Acid – 100ml

Allow the dye to dissolve and adjust the pH to 2.4 by using 1 M of sodium hydroxide. Store in a dark bottle to prevent reaction of dye with the light. This reagent is stable at room temperature for 6 months.

2. 3% Glacial Acetic Acid
· Glacial Acetic Acid – 6ml
· Type II water (DI Water) – 200ml

Store at room temperature.This reagent is stable for 1 year.

Staining procedures:
1. Heat the slide containing the section on the hotplate for 3 minutes
2. Dewax the section using the auto-stainer (machine)
3. Bring section to water to prevent section from drying up
4. Rinse the section using 3% aqueous Glacial Acidic Acid solution
5. Stain in 1% Alcian Blue in 3% aqueous Glacial Acidic Acid for 20 minutes
6. Rinse in 3% aqueous Glacial Acidic Acid solution before rinsing in water
7. Counterstain with Nuclear Fast Red for 5 minutes
8. Rinse the section with water
9. Dehydrate the section using graded alcohol from 95% to absolute alcohol
10. Clear the section using 3 changes of xylene
11. Mount the slide with Depex

Results:
· Acid mucins – Blue
· Nuclei – Reddish pink

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What is mesothelioma?

· Cancer of mesothelium
· Mesothelium is a thin membrane that surrounds most of our internal organs for protection purpose. The
membrane consists of 2 layers; one on the organ, another forms a sac around it. In the sac, there is a fluid
produced by the mesothelium to reduce friction as our organs glide against one another or against our
body structure such as the rib cage.

What is atherosclerosis?

· Hardening or arteries
· This is due to the buildup of deposits on the walls of the arteries. Common deposits are fatty substances,
cholesterol, calcium and cellular waste products.
· Buildup of deposits is known as plague and can rupture the arteries, causing the blood clots to obstruct
blood flow or travel to other parts of the body, resulting in heart attack, stroke etc.

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Masson Trichome (MT)

Function:
· Stains muscle, fibrin and collagen

Principle:
· A section is first treated with Masson Ponceau Fuchsin (small molecule anionic dye) before treated with
phosphomolybdic acid or phosphotungistic acid solution
· Phosphomolybdic acid or phosphotungistic acid solution will compete woth the dye to gain acess to the
collagen, at the same time, expelling the dye in the process
· The collagen is then treated with a larger molecule green or blue dye

Control used:
· Liver tissue, skin or muscle

Fixative used:
· 10% Neutral Buffered Formalin

Chemicals required:
· Haemotoxylin
· 4% aqueous phosphomolybdic acid
· Masson’s Light Green
· Ponceau 2 R
· Acid Fuchsin
· Glacial Acetic Acid

Reagent preparation:

1. Masson Ponceau Fuchsin
· Ponceau 2 R – 3.5g
· Acid Fuchsin – 1.75g
· Glacial Acetic Acid – 5ml
· Type II water (DI Water) –500ml

This reagent is stable at room temperature for 6 months.

2. 2% Phosphomolybdic- tungstic Acid
· Phosphomolybdic Acid – 4g
· Phosphotungistic Acid – 4g
· Type II water (DI Water) – 200ml

Dissolve in water and heat it at 50-60oC overnight by putting it in the oven.
Filter and store at room temperature.

3. 1% Acetic Acid
· Glacial Acetic Acid – 2ml
· Type II water (DI Water) – 98ml
Mix before use. Stable at room temperature for 6 months.

Staining procedures:
1. Heat the slide containing the section on the hotplate for 3 minutes
2. Dewax the section using the auto-stainer (machine)
3. Bring section to water to prevent section from drying up
4. Post fix the section in Bouin’s solution for 30 minutes at 56oC
5. Stain nuclei with Weigert’s iron haemotoxylin for 10 minutes
6. Wash in tap water and blue for 5 minutes
7. Stain in Masson’d Ponceau-Fuchsin for 5 minutes
8. Wash in 1% Acetic Acid
9. Stain in Masson’s Light Green for 30 seconds to 2 minutes, until collagen is green. In order to
control intensity of the stain, it is better to stain for 1 minute first, then rinse in acid water before examining
under the microscope.
10. Rinse in 1% Acid Alcohol
11. Dehydrate the section using graded alcohol from 95% to absolute alcohol
12. Clear the section using 3 changes of xylene
13. Mount the slide with Depex

Results:
· Nuclei – Blue black
· Cytoplasm – Light red
· Muscle – Dark red
· Red cells – Bright red
· Hyaline & fibrin – Bright red
· Collagen & mucin – Green

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Cheers!

Ting Ying Chee
TG01